CN100390264C

CN100390264C - A device.

Info

Publication number: CN100390264C
Application number: CNB018125867A
Authority: CN
Inventors: I·M·S·S·阿布亚达耶
Original assignee: Tristem Trading Cyprus Ltd
Current assignee: Tristem Trading Cyprus Ltd
Priority date: 2000-05-10
Filing date: 2001-05-10
Publication date: 2008-05-28
Anticipated expiration: 2021-05-10
Also published as: ZA200208769B; CN1774499A; CN101538551A; CN101386839A

Abstract

A device for preparing an undifferentiated cell, the device comprises means for contacting a more committed cell with an agent that causes the more committed cell to retrodifferentiate into an undifferentiated cell.

Description

A kind of equipment

Technical field of the present invention

The present invention relates to a kind of technology.More specifically, the present invention relates to a kind of equipment for preparing undifferentiated cell.Especially, but not ad hoc, the present invention relates to a kind of equipment from the cell preparation undifferentiated cell of more finalizing the design.On the other hand, the present invention also relates to a kind of method that forms undifferentiated cell.This equipment also can carry out long-range the contact with the producer/sellers/producer/call center/service centre etc., for example long-rangely orders novel agent and/or validation operation or is correctly carried out.Therefore, the present invention also relates to center on this equipment commerce method with and uses thereof, for example carry out long-range the contact with the producer/sellers/producer/call center/service centre etc., the producer/sellers/producer/call center/service centre etc. (is for example answered this contact, receive and fill in order, receive and/or handle and/or data/information and/or its corrigendum of answer) about operating.

Background of invention

Differentiation is a kind of process, finalizes the design gradually by the 26S Proteasome Structure and Function of this process cell, to produce more special cell, for example forms T cell or B cell from prematurity hematopoiesis precursor.Therefore, when cell becomes when more finalizing the design, they become more special.In most cell types, cytodifferentiation is the method that causes the single channel of final noble cells.Yet, although some cell types keep not dividing in whole life, and not replaced, and many cell types continue division in the whole life of organism, and upgrade.This may be simple division (as a liver cell), perhaps as in hematopoietic cell and epithelial cell, is to be divided by undifferentiated relatively stem cell, carries out subsequently irreversible differentiation by one of daughter cell then.Yet all these processes have a common trait: cell or keep its differentiation state, more differentiation perhaps becomes.They can not become not differentiation or even more not break up.

Reverse differentiation (retrodifferentiation) is a kind of process, and the 26S Proteasome Structure and Function by this process cell changes gradually, to produce more not specified cell.Some cell responses are in tissue injury and naturally in vivo carry out limited reverse differentiation.For example, observed when liver degenerates, liver cell reverts to expression of enzymes pattern (Curtin andSnell, 1983, Br.J.Cancer, the Vol.48 with fetus enzyme pattern similarity; 495-505).

Shown among the WO96/23870 and can handle,, comprised stem cell to be that they become undifferentiated cell to noble cells.These undifferentiated cells can be bred, and produce the reverse differentiation offspring of same clone or any other clone.For the hematopoietic cell of reverse differentiation, these stem cells are polyenergic, and they can produce a plurality of clones.The initiative discovery of among the WO96/23870 this is unexpected fully.

The clinical meaning of this discovery is huge.For human patients, stem cell is extremely difficult to be obtained.They generally are to obtain from umbilical cord tissue, marrow or blood, and their amount is very little at these positions.Yet, the invention provides and a kind ofly produce the equipment of stem cell from committed cell more, particularly produce the equipment of stem cell by reverse process of differentiation.

US 6,087, and 168 disclose and a kind of epithelial cell changeed the method be divided into neuronal cell, wherein epithelial cell are dedifferented or reverse differentiation with suitable substratum.Same Lake JAet al Journal of Cell Science 113,556-566 (2000) and Rathjen Jet al Journal of Cell Science 112,601-612 (1999) discloses the embryo has been done reverse early stage primitive ectoderm sample (EPL) cell that is divided into of (ES) cell, and this reacts on two kinds of separable factors and causes.

Summary of the invention

Aspect wideer, a kind of equipment for preparing undifferentiated cell is provided, wherein this equipment comprises the more reverse instrument (means) that is divided into undifferentiated cell of committed cell that allows.

Prior art file (as US 6,087,168, Lake et al or Rathjen et al) all openly is not applicable to the equipment of preparation undifferentiated cell.Although the preparation undifferentiated cell can not used equipment of the present invention and finished by those skilled in the art, to carry out the finished product that this program obtains inconsistent easily at every turn, and this result depends on the people's who carries out this program technology and experience.In addition, artificial preparation undifferentiated cell desired " processing " is chronic, is not economical for the laboratory therefore.

In a particular, a kind of relative number destination device that forms undifferentiated cell and/or increase undifferentiated cell in comprising committed cell's cell mass is provided, this equipment comprises a chamber (chamber), the cell mass that will comprise the committed cell is introduced the instrument of described chamber, can make the reverse reverse differentiation instrument that is divided into undifferentiated cell of committed cell introduce the instrument of described chamber, and under the condition that described reverse differentiation instrument exists, hatch described committed cell's the instrument of hatching, thereby make the reverse undifferentiated cell that is divided into of committed cell.

In another particular, a kind of relative number destination device that forms undifferentiated cell and/or increase undifferentiated cell in comprising committed cell's cell mass is provided, this equipment comprises a chamber, the cell mass that will comprise the committed cell is introduced the instrument of described chamber, to cause the reverse reagent that is divided into undifferentiated cell of committed cell to introduce the instrument of described chamber, and the instrument of hatching of hatching described reagent and described committed cell, thereby make the reverse undifferentiated cell that is divided into of committed cell.

Preferably, this reagent relates to the acceptor of catching, discerning or present of (engage) mediation committed cell surface antigen.More preferably, this receptor is MHC I class antigen or MHC II class antigen, as is selected from the MHC I class antigen of relevant (the HLA)-A acceptor of human leukocyte, HLA-B acceptor, HLA-C acceptor, HLA-E acceptor, HLA-F acceptor or HLA-G acceptor or is selected from the MHC II class antigen of HLA-DM acceptor, HLA-DP acceptor, HLA-DQ acceptor or HLA-DR acceptor.

Suitably, this receptor can contain the β chain with homology zone.Preferably, this receptor can contain the homologous region of the β chain of HLA-DR at least.

Usually, the committed cell is a noble cells.Preferably, the committed cell is the hematopoietic cell of typing, is preferably selected from the T cell colony and forms cell (CFC-T cell), B cell colony formation cell (CFC-B cell), eosinophil colony forming cell (CFC-Eosin cell), basophilic cell colony forming cell (CFC-Bas cell), granulocyte/monocyte colony forming cell (CFC-GM cell), megakaryocyte colony forming cell (CFC-MEG cell), red corpuscle explosion type colony forming cell (BFC-E cell), red corpuscle colony forming cell (CFC-E cell), T cell and B cell.

In a preferred embodiment of the invention, more the committed cell is not a cancer cells.In another preferred embodiment of the present invention, this reagent can not promote the cancer growth neither carcinogenic.

In a preferred embodiment, this reagent is the antibody of acceptor, as the monoclonal antibody of acceptor.Special example comprises CR3/43 and TAL.1B5 monoclonal antibody.

In a preferred embodiment, this reagent is regulated the mhc gene expression.Suitably, this reagent can be regulated MHC I class ⁺And/or MHC II class ⁺Express.

This reagent preferably with biological response modifier, unite use as alkylating agent, for example be or comprise the alkylating agent of endoxan.

Preferred undifferentiated cell comprises stem cell antigen.In a preferred embodiment, undifferentiated cell is selected from embryonic stem cell, multipotential stem cell, lymphoid stem cells, hemopoietic stem cell, neuronal stem cell, epithelial stem cell, mescenchymal stem cell, endothelial stem cell and myeloid stem cell.Preferably, undifferentiated cell is characterised in that following one or more cell surface marker symbol: CD34 ⁺, HLA-DR ^-, CD38 ^-, CD117, AC133, CD90 and/or low CD45 (CD45 low).More preferably, undifferentiated cell is CD34 ⁺And CD38 ^-, even CD34 more preferably ⁺, CD38, HLA-DR ^-With low CD45.

Therefore, in a preferred embodiment of the invention, the present invention also provides a kind of increasing in comprising committed cell's cell mass to have cell surface marker symbol CD34 ⁺And/or CD38 ^-And/or HLA-DR ^-And/or the relative number destination device of the cell of low CD45 and/or CD90 and/or CD117 and/or AC133, this equipment comprises:

(i) chamber,

The cell mass that (ii) will comprise the committed cell is introduced the instrument of described chamber

The reagent that (iii) operability is related to described committed cell is introduced the instrument of described chamber; With

(iv) operability is hatched the described committed cell's who is related to by described reagent the instrument of hatching, and makes owing to described relating to increases CD34 ⁺And/or CD38 ^-And/or HLA-DR ^-And/or the relative number of low CD45 and/or CD90 and/or CD117 and/or AC133 cell.

Equipment of the present invention can randomly further comprise the described undifferentiated cell of enrichment, remove this reagent and/or reclaim the purifying and the separating tool of described undifferentiated cell from the cell mass that changes.Preferably, tools for purification comprises and identifies and to be present in the cell surface marker on undifferentiated cell surface or to be present in the committed cell surface but not to be present in the cell surface marker on undifferentiated cell surface substantially.Suitably, tools for purification can be utilized for example antibody of anti-cell surface markers.The example of suitable mark comprises CD34, CD45 and HLA-DR.Only illustrate, suitable tools for purification is a CliniMACs/Isolex CD34+ purification system.In another preferred embodiment of the present invention, this purifying or separating tool can be randomly provide as instrument independently.

Equipment of the present invention can randomly further comprise negative selection in upstream and/or cell enrichment instrument.In addition, can before inserting described equipment, optional pair cell group bear selection and/or cell enrichment.

In another preferred embodiment, undifferentiated cell of the present invention is CD34 ⁺CD45 ^-With there not being hematopoietic cell is the cell of mark.

More Fen Hua cell can with primary committed cell homology or non-homology.

Therefore, except producing undifferentiated cell, equipment of the present invention can be used for the cell transformation of a system is another cell that is.

Therefore, another aspect of the present invention provides the cell mass of the typing hematopoietic cell that will comprise a kind of hematopoiesis system to induce equipment into the cell of another hematopoiesis system, and this equipment comprises

(i) chamber,

The antigenic reagent of catching, discerning or present that (iii) operability is related to described typing hematopoietic cell surface is introduced the instrument of described chamber; With

(iv) operability is hatched the described committed cell's who is related to by described reagent the instrument of hatching, and makes them owing to described relating to becomes the cell that another kind of hematopoiesis is.

Preferably, the hematopoietic cell of described typing belongs to B clone, and becomes the cell of the another kind of hematopoiesis system that is selected from T clone and myelocytic series.

The undifferentiated cell that is produced by equipment of the present invention can be used to make the medicine for the treatment of immunologic derangement or disease.Equally, the committed cell of the method according to this invention generation can be used to make the medicine of treatment immunologic derangement or disease.

The present invention is highly useful, because can use these undifferentiated cell bodies interior or external then easily from more preparing undifferentiated cell the committed cell now, or the two be in conjunction with the preparation medicine, so that the treatment disease.

Another advantage of the present invention is the cell that it also can be fixed to again typing with the undifferentiated cell that this equipment will be prepared by reverse differentiation, as new noble cells, its purpose is for correcting or removing original more committed cell or correction or remove its product.For example, the cell that can produce typing again with undifferentiated cell is as covering the cell of alveolar, can substitute or repair thus destroy lung tissue or make its morbific mechanism (see Le Page, New Scientist 19 December2000, p20).

Noun " Ding Xing cell again " expression derives from the cell of undifferentiated cell, promptly new more committed cell.The expression of " more finalizing the design " is more broken up, and therefore can determine according to known cytodifferentiation approach and stage.

Most of undifferentiated cells and noble cells comprise main tissue compatible type mixture (MHC) I class antigen and/or II class antigen.If these antigens combine with those cells, they are called the I class so ⁺With the II class ⁺Cell.Preferably, more the committed cell can be divided into MHC I class again ⁺And/or MHC II class ⁺Undifferentiated cell.

Preferably, more the committed cell can reversely be divided into the undifferentiated cell that comprises stem cell antigen.

Preferably, more the committed cell can reversely be divided into CD34 ⁺Undifferentiated cell.

Preferably, more the committed cell can reversely be divided into the lymph hemopoietic progenitor cell.

Preferably, more the committed cell can reversely be divided into multipotential stem cell.

Preferably, described being increased in 24 hours taken place in preferred 4-8 hour (any like this change can not be explained by cell proliferation fully).

Usually, the mensuration that changes of undifferentiated cell number is to change by the number of cell that monitoring has a characteristic cell surface marker of undifferentiated cell to carry out.The example of suitable cell surface marker comprises CD34 ⁺, HLA-DR ^-, CD38 ^-, CD117, AC133, CD90 and/or low CD45.As selecting or replenish, can monitor the minimizing of number of the cell of characteristic cell surface marker with noble cells rather than undifferentiated cell.

Suitably, equipment of the present invention can randomly further comprise the trace tool of the number change of the cell of monitoring the characteristic cell surface marker with undifferentiated cell.

The preferred committed cell who is used to measure is the hematopoietic cell of typing, as is selected from the CFC-T cell, CFC-B cell, CFC-Eosin cell, the CFC-Bas cell, CFC-GM cell, CFC-MEG cell, BFC-E cell, the CFC-E cell, T cell and B cell, the more preferably cell of B cell.

Evaluation method selecting optimal equipment of the present invention further comprise one or more, preferably two or more, more preferably three kinds or more, more preferably four kinds or how following feature:

(i) survey instrument of measurement cell mass volume,

(ii) carry out cell counting and measure the count tool (time carried out microminiaturized Coulter-counter or other suitable cell counter) if necessary of the cell concn of cell mass thus,

(iii) the cell mass of a certain amount of (as predetermined amount) is transferred to means of transferring the chamber from storage vessel, this means of transferring can be chosen wantonly and comprise for example pump,

(iv) calculate the computational tool of the volume that joins the reagent in the chamber, the volume of this reagent depends on the volume and the cell concn of cell mass,

(v) with the agent transfer of certain volume (as the volume that calculates) the another kind of means of transferring in the chamber, this another kind means of transferring can be chosen wantonly and for example comprise by motor-driven syringes such as stepper-motors,

(vi) be used for controlling the carbonic acid gas control tool (for example as a part of hatching instrument) of chamber gas concentration lwevel,

(vii) be used for controlling the temperature control tool (for example as a part of hatching instrument) of chamber temperature,

(viii) be used for the cell mass of mixing chamber and the mixing tool of reagent (for example as a part of hatching instrument),

(ix) be used for the stage of hatching is carried out the timing tool (for example as hatching the part of instrument) of timing, and optional being used for show the show tools of remaining time and/or the prompting instrument of reminding the user to finish in the stage of hatching to the user,

(x) be used for from the chamber harvested cell, particularly from chamber, gather in the crops the harvesting tool of undifferentiated cell,

(xi) take out the cell mass the comprise undifferentiated cell taking-up instrument to the storage vessel from chamber, the taking-up instrument can comprise, for example pump; With

(xii) sealing tool of sealed storage container wherein contains the cell mass that comprises undifferentiated cell.

Suitably, in equipment of the present invention, means of transferring (iii) can with the cell mass of a certain amount of (as predetermined amount) directly from chamber as described in the patient transfers to (promptly not needing storage vessel) and/or taking-up instrument (xi) can from chamber, directly take out comprise undifferentiated cell cell mass to patient's (promptly not needing storage vessel).

Suitably, means of transferring (iii) can comprise peristaltic pump, and this pump is transferred to cell mass in the chamber from storage vessel by interconnected instrument such as pipeline.Interconnected instrument preferably passes through Coulter-counter or other suitable cell counter (mentioning) in (ii).By this way, can the process of cell mass being transferred to chamber from storage vessel, calculate by pair cell group's cell concn.

Preferably, more than (iii) and the storage vessel (xi) mentioned be reservoir bag, preferably disposable.Storage vessel (iii) is preferably preferably different with the storage vessel of (xi), and the former is the input storage vessel, and the latter is the output storage vessel.

Preferably, (reservoir that v) comprises a kind of reagent is so that guarantee whenever all to have enough reagent for another kind of means of transferring.When means of transferring is syringe, can provide reservoir with another syringe, when a syringe emptying, another syringe begins to supply reagent like this.

Preferably, the carbonic acid gas control tool that is used for controlling the chamber gas concentration lwevel comprises a valve, and its allows to introduce the carbonic acid gas of predetermined amount from gas cylinder.The carbonic acid gas of predetermined amount is to calculate with the measurement volumes of the cell mass of known air volume in chamber, pipeline and the output storage vessel and input storage vessel.Suitably, by a port carbonic acid gas is introduced in the chamber, this port is identical with the port that adding reagent place passes through, and in this way, carbonic acid gas can be used for all remaining reagent is delivered to port and equipment on every side.Except that above-mentioned, the carbonic acid gas control tool can further contain inflatable air bag, to hold the unnecessary gas volume that produces by with extra carbonic acid gas drawing-in system.In addition, in case the emptying of input storage vessel just can be used as air bag.Preferably, the final concentration that produces in chamber of the carbonic acid gas of introducing is 5%.

(vii) can suitably comprise heating tool, this heating tool can provide by the hot plate that for example is positioned under the chamber temperature control tool.In addition, heating tool can be by being adjacent to chamber or its a part of hot water jacket provides.As a reference, the temperature in the chamber is controlled at about 25 ℃-Yue 37 ℃.

(viii) preferably comprise at least one vane-type arm, it slowly rotates with the reagent in mixed cellularity group and the chamber mixing tool.Also can select to use magnetic stirrer.Select as another kind, can carry out mechanical stirring to chamber, for example by shaking gently.The advantage that use comprises the mixing tool of at least one vane-type arm is that this arm also can be designed as the curette in the cell harvesting process, and promptly when this arm slowly rotated, the cell that is attached to chamber surfaces was removed gently.Advantageously, this mixing tool can operability be realized cyclotron motion.

Taking-up instrument (xi) preferably comprises a peristaltic pump.Advantageously, the taking-up instrument is by pulling out the cell mass that comprises undifferentiated cell at the output port of cavity bottom, and by interconnected instrument, for example pipeline enters the output storage vessel.

As the selection of taking out instrument or replenish, can before taking out cell mass, in chamber, add dissociated ion sequestering agent and/or sequestrant, for example antithrombotics such as EDTA.In chamber, add this ion sequestering agent and/or sequestrant and cause stem cell colonies to be separated into the suspension of individual cells, and therefore promote from chamber, to take out cell by washer.

Sealing tool (xii) preferably comprises a heat sealer, and this heat sealer is that two portions by with container force together to closing of output storage vessel, and the two portions that maybe will be positioned at the interconnected instrument before this container force together, up to sealing fully.For example, heat sealer may produce the wide strip of paper used for sealing of about 2cm, and heat sealer is partly cut off strip of paper used for sealing then therebetween.

Each part of the present invention preferably can independently be controlled by central computer system.Advantageously, computer system can realize calculating according to these input signals from a part of receiving inputted signal of this equipment, and output signal is sent to identical or another part of this equipment.

Equipment of the present invention has some advantages, has obtained consistent result when particularly this equipment has guaranteed each steering routine, and does not waste expensive reagent.In addition, can programme to this equipment, so as when reverse differentiation finishes, to remind the user and/or finish before the demonstration time remain.If to carry out this task, this equipment can impel the user to buy novel agent to its programming.In other words, therefore use and margin that this equipment can monitoring reagent impel the user to buy when the reagent of laying in is reduced to threshold value when following.

Equipment of the present invention can further comprise a plurality of chambers, and each is used for generation and is fixed to different clones, as the undifferentiated cell of muscle, hair, neurone etc.Like this, can produce the undifferentiated cell that is fixed to different clones simultaneously.Preferably, when using a plurality of chamber, each chamber has independently output port and independently exports storage vessel.

Can be by each chamber being used the plastics (plastics) of different treatment or in each chamber, adding different chemical substances and in equipment of the present invention, control clone.

One or more compositions of this equipment are preferably disposable.For example, one or more compositions below can be disposable: chamber, input storage vessel, output storage vessel, the interconnected instrument that connects storage vessel and chamber and means of transferring are (v).Suitably, disposable composition can be used as module (cartridge) and is used to insert this equipment.Only be to illustrate as an example, module can comprise first blood bag (input storage vessel), a chamber and second (output) blood bag, and its middle chamber is connected with first each with second blood bag by pipeline (interconnected instrument).Suitably, this equipment can be considered to a kind of partial fixing and the disposable system of part.

Preferably, the chamber of the equipment that contacts with cell mass and/or other compositions are can not produce the plastics of other forms of cell transformation body with USP VI class material, polycarbonate patch or other.

The cell mass that preferably comprises is a blood.Preferred reagent is antibody.

On the other hand, this provides a kind of method for preparing undifferentiated cell clearly, this method comprises and is divided into undifferentiated cell with committed cell more is reverse that wherein more committed cell's reverse differentiation betides more committed cell in buffy coat (buffy coat) blood sample or that come from the buffy coat blood sample.

On the other hand, this provides clearly a kind of method for preparing undifferentiated cell, this method to comprise to make more committed cell in the buffy coat blood sample or that come from the buffy coat blood sample that the reverse reagent that is divided into undifferentiated cell of committed cell contacts with causing more.

In this clearly demarcated a kind of preferred embodiment, more the committed cell is not a cancer cells.In this clearly demarcated another kind of preferred embodiment, this reagent is not carcinogenic, can not promote the cancer growth.

The cell of typing preferably breaks up.The hematopoietic cell that the cell of typing is preferably finalized the design, as be selected from the CFC-T cell, CFC-B cell, CFC-Eosin cell, CFC-Bas cell, CFC-GM cell, CFC-MEG cell, BFC-E cell, CFC-E cell, T cell and B cell, more preferably B cell.

Undifferentiated cell is preferably selected from multipotential stem cell, hemopoietic stem cell, neuronal stem cell, epithelial stem cell, mescenchymal stem cell and embryonic stem cell.Preferably, undifferentiated stem cell is characterised in that following one or more cell surface marker symbols CD34 ⁺, HLA-DR ^-, CD38 ^-, CD117, AC133, CD90 and/or low CD45.More preferably, undifferentiated cell is CD34 ⁺And CD38 ^-, even CD34 more preferably ⁺, CD38 ^-, HLA-DR ^-With low CD45.

Suitably, undifferentiated cell is a MHC I class ⁺And/or MHC II class ⁺Cell.

Described reagent preferably relates to the acceptor of catching, discerning or present of mediation committed cell surface antigen.More preferably, this receptor is MHC I class antigen or MHC II class antigen, as is selected from the I class antigen of relevant (the HLA)-A acceptor of human leucocyte, HLA-B acceptor, HLA-C acceptor, HLA-E acceptor, HLA-F acceptor or HLA-G acceptor or is selected from the II class antigen of HLA-DM acceptor, HLA-DP acceptor, HLA-DQ acceptor or HLA-DR acceptor.

Suitably, this receptor can comprise the β chain with homologous region.In one embodiment, this receptor can comprise the homologous region of HLA-DR β chain at least.

In a preferred embodiment, this reagent is the antibody of acceptor, as the monoclonal antibody of acceptor.Concrete example comprises CR3/43 and monoclonal antibody TAL.1B5.

Suitably, this reagent can be regulated mhc gene and express, for example MHC I class ⁺And/or mhc class ii ⁺Express.

In this another clearly demarcated preferred embodiment, provide in a kind of increase buffy coat blood sample to have cell surface marker symbol CD34 ⁺And/or HLA-DR ^-And/or CD38 ^-And/or the relative number purpose method of the cell of CD117 and/or AC133 and/or CD90 and/or low CD45, this method comprises that the more committed cell who makes in the buffy coat blood sample contacts with the reagent that operability relates to this committed cell, this relating to, cause CD34 ⁺And/or HLA-DR ^-And/or CD38 ^-And/or the relative number of CD117 and/or AC133 and/or CD90 and/or low CD45 cell increases.

On the other hand, this provides a kind of method for preparing undifferentiated cell clearly, this method comprises one or more noble cellss and effective reverse differentiation tool in contact of changing the normal differentiation cell proportion in the described cell mass that make in the cell mass, causes the reverse undifferentiated cell that is divided into of one or more described noble cellss thus.

On the other hand, this provides the reverse differentiation instrument of the normal differentiation cell proportion among the transition cell group to realize the reverse purposes that is divided into undifferentiated cell of one or more described noble cellss clearly.

On the other hand, this provides a kind of method for preparing undifferentiated cell clearly, this method comprises the reverse undifferentiated cell that is divided into of the noble cells in the cell mass, the environment that wherein comprises the cell mass that contains one or more noble cellss is second kind of environment from first kind of environment change, the dissociated ion concentration of wherein said second kind of environment has been carried out effective modification with first kind of environmental facies ratio, causes the reverse undifferentiated cell that is divided into of one or more described noble cellss thus.

On the other hand.This provides a kind of method for preparing undifferentiated cell clearly, this method comprises one or more noble cellss in the cell mass and the reverse differentiation tool in contact of effectively changing the normal differentiation cell proportion, culturing cell group in not containing first kind of masked environment of ion or ion, and be second kind of environment with first kind of environment change, wherein the ionic concn that exists in second kind of environment has been carried out effective modification with first kind of environmental facies ratio, causes the reverse undifferentiated cell that is divided into of one or more described noble cellss thus.

Preferred reverse differentiation instrument is any ratio destructive instrument that causes normal differentiation cell in the cell mass, is called the negative selection in the cell mass herein, the destruction that causes the ratio of normal differentiation cell in the cell mass thus.

Reverse differentiation instrument can be, for example, and following any one or multiple: antibody (pure and link coupled (promptly being incorporated into fixed and free part) as magnetic, glass or polystyrene bead; Histopaque, LymphoPrep (Sigma)) or other be used for any density gradient substratum according to the cell density isolated cell; Or dextran (for example causing erythroprecipitin).Other cause suitable instruments of cell conversion see Vettese-Dadey (The Scientist, Sep 13 1999,13 (18): 21).

Dissociated ion concentration in second kind of environment and first kind of environmental facies are than increasing to some extent.

More preferably, the relative dissociated ion concentration of first and second kind environment increases, and promptly the dissociated ion concentration of second kind of environment increases.Preferably, the increase of dissociated ion concentration enough causes the reverse undifferentiated cell that is divided into of one or more noble cellss relatively.As just illustrating, with cell from the media transfer that contains 5mM EDTA (a kind of dissociated ion sequestering agent and/or sequestrant) to the another kind of substratum that contains still less or do not contain EDTA, cause the increase of dissociated ion concentration (as calcium ion concn), this increase is enough to cause reverse differentiation.

Preferred dissociated ion is a positively charged ion.

Preferred dissociated ion is I family or II family metal.

Preferred cationic is calcium ion and/or magnesium ion.

Suitably, the dissociated ion concentration of environment can be by modifying with the agent treated that can change dissociated ion concentration in the environment relatively.

Therefore, in a kind of preferred embodiment, this provides a kind of method for preparing undifferentiated cell clearly, this method comprises the reverse undifferentiated cell that is divided into of the noble cells in the cell mass, the environment that wherein contains this cell mass is modified by using the agent treated that can change dissociated ion concentration in the environment relatively, thereby realizes second kind of environment.

For example, first kind of environment can be handled with one or more dissociated ion sequestering agents, with its removal or minimizing, realization has second kind of environment of the dissociated ion concentration of relative increase thus subsequently, thereby realizes the reverse differentiation of one or more noble cellss in the cell mass.In other words, for example can remove or reduce sequestering agent in the following manner: from first kind of environment, remove the part of sequestering agent or sequestering agent by physics or chemical process, perhaps cell mass is transferred to do not have this dissociated ion sequestering agent or with first kind of second kind of lower environment of environmental facies specific ionization ion sequestering agent concentration.

Under any circumstance, the dissociated ion concentration of second kind of environment increases to some extent with first kind of environmental facies ratio, thereby has realized the reverse differentiation of one or more noble cellss in the cell mass.

As selection, can contain the lower concentration dissociated ion or not contain culturing cell group in first kind of environment of dissociated ion, then cell mass is transferred to second kind of environment, or regulate first kind of environment, make it become second kind of environment, described second kind of environment contains ion, or with first kind of environmental facies ratio, ionic concn is higher, thereby has realized the reverse differentiation of one or more noble cellss in the cell mass.The term of herein using " low " comprises with the final concentration of second kind of environment to be compared, and has the environment of low relatively dissociated ion initial concentration.

Therefore, in a kind of preferred embodiment, this provides a kind of method for preparing undifferentiated cell clearly, this method comprises the reverse undifferentiated cell that is divided into of the noble cells in the cell mass, the environment that wherein contains the described cell mass that comprises one or more noble cellss never free calcium ions or magnesium ion or calcium ion or first kind of low environment change of magnesium ion concentration is second kind of environment, described second kind of environment calcium ions or magnesium ion, or with first kind of environmental facies ratio, calcium ion or magnesium ion concentration are higher, thereby have realized the reverse differentiation of one or more noble cellss in the cell mass.

Without wishing to be bound by theory, think that it is colony (for example, carrying out homotypic aggregation) that the increase of change, particularly ionic concn of ionic concn causes the cell aggregation in the cell mass, these intercellular physics contacts can be induced their reverse differentiation.

Sequestering agent is preferably ion chelating agent.

Sequestering agent preferably comprises amine and carboxyl simultaneously.

Preferably, sequestering agent comprises a plurality of-N (CH ₂CO ₂H) _nGroup, wherein n=1 or n=2.

Suitably, sequestering agent can be selected from following one or more: EDTA, heparin, EGTA, DTPA, sequestrant that trisodium citrate is similar with other and/or antithrombotics.

Suitably, can add the sequestering agent of enough high densitys, feasible it is removed can cause reverse differentiation.Usually, after being removed, it enough cause the concentration of sequestering agent of reverse differentiation for more than or equal to about 2mM.

In this clearly demarcated a kind of preferred embodiment, more the committed cell is not a cancer cells.In another kind of preferred embodiment, this reagent can not promote growth of cancer cells neither carcinogenic.

Undifferentiated cell is preferably selected from multipotential stem cell, hemopoietic stem cell, neuronal stem cell, epithelial stem cell, mescenchymal stem cell and embryonic stem cell.Preferably, undifferentiated cell is characterised in that following one or more cell surface marker symbols CD34 ⁺, HLA-DR ^-, CD38 ^-, CD117, AC133, CD90 and/or low CD45.More preferably, undifferentiated cell is CD34 ⁺And CD38 ^-, even CD34 more preferably ⁺, CD38 ^-, HLA-DR ^-With low CD45.

On the other hand, this provides a kind of method for preparing undifferentiated cell clearly, this method comprises the reverse undifferentiated cell that is divided into of the noble cells in the cell mass, wherein comprising the described environment that contains one or more noble cellss is second kind of environment from first kind of environment change, the dissociated ion concentration of wherein said second kind of environment has been carried out effective modification with first kind of environmental facies ratio, causes the reverse undifferentiated cell that is divided into of one or more described noble cellss thus.

In one embodiment, the cell mass that comprises the committed cell is in the buffy coat blood sample or comes from the buffy coat blood sample.

Advantageously, can effectively cultivate the red corpuscle progenitor cell with this clearly demarcated a kind of method, to produce red corpuscle, this red corpuscle can be used for for example shortage of supplemental blood supply.Suitably, this clearly demarcated a kind of method can be used for generation and be used for the aborning megalokaryocyte of thrombocyte.

For this can clearly be understood and implement easily clearly, will be with reference to the following drawings as an example.

Fig. 1 represents the skeleton view that well-behaved exposed installation is equipped with, and its front panel is opened, and illustration represents to break away from the sectional elevation of the equipment of its environment;

The skeleton view of the equipment of Fig. 2 presentation graphs 1, its front panel is closed;

Fig. 3 represents the photo of hemocyte;

Fig. 4 represents that LSCs is differentiated to form the chart of T cell and B cell;

Fig. 5 represents that MSCs is differentiated to form eosinophilic granulocyte, basophilic granulocyte, neutrophil leucocyte, megalokaryocyte, monocyte, red corpuscle, granulocyte, mastocyte, NK cell and lymphocyte;

Fig. 6 represents the lymph hemopoietic progenitor cell.

Fig. 7 represents with CD45 after the CR3143 antibody treatment ^-CD14 ^-The outward appearance of cell;

Fig. 8 represents to break up the microphotograph of B cell;

Fig. 9 represents the microphotograph by the reverse undifferentiated cell that is differentiated to form of B cell;

Figure 10 represents the microphotograph with the same undifferentiated cell of Fig. 9; But magnification is lower;

Figure 11 represents to break up the microphotograph of B cell;

Figure 12 represents the microphotograph by the reverse undifferentiated cell that is differentiated to form of B cell;

Figure 13 represents to form the granulocytic microphotograph of differentiation from the undifferentiated cell identical with Figure 12;

Figure 14 represents the microphotograph of two different amplification of the blood sample that is untreated that obtains from BCLL patient;

Figure 15 represents the microphotograph of treated blood sample;

Figure 16 and 17 is illustrated in and handles time lapse microphotograph in the blood sample process;

Figure 18 represents southern blotting technique;

Figure 19 represents the another kind of southern blotting technique with the acquisition of B-CLL peripheral blood of patients cell;

Figure 20 represents to increase CD34 in the cell mass with three kinds of reagent ⁺The relative number of cell;

Figure 21 represents to measure with the stem cell colonies of anti-phase bright-field microscope observation;

Figure 22 represents the attached cell with anti-phase bright-field microscope observation; With

Figure 23 represents two rest images obtaining with time lag video recording in the CR3/43mab treatments B cell processes.

Detailed Description Of The Invention

I equipment

This equipment carries out integral body with reference number 1 to be described, and comprises a shell 2 with front panel 3, and front panel 3 can be opened, and allows the inside of the equipment that reaches 1.Prop up and hang with an input storage vessel on the tenaculum 5, promptly the blood bag 4.Prop up tenaculum 5 and formed a kind of electrobalance (not shown), can operate, be used for blood bag 4 is weighed.

This equipment further comprises a chamber 6, and this chamber 6 is interconnected by pipeline 7 with input blood bag 4.Pipeline 7 comprises a transparent window 8, and this window is positioned at the Coulter-counter flow cell.Can operate a peristaltic pump (not shown), pull out blood, enter chamber 6 through counter stream pond 9 from input blood bag 4.

Syringe

10 and 11 contains antigen, all is to drive with the stepper-motor (not shown).

Syringe

10,11 continues to maintain 4 ℃ the insulation pal of locking and pastes in " refrigerator " 12.It is in order to guarantee that this equipment always has enough blood and supplies that two

syringes

10 and 11 are provided.Use microbiotic and disposable 0.2 μ m filter membrane (generally becoming reference number 13) to keep the sterility of

syringe

10 and 11.

This equipment also comprises a carbonic acid gas inlet 14, and it instructs carbonic acid gas to enter chamber 6 by pipeline 15 from the gas cylinder (not shown).

Syringe

10 and 11 also forms the fluid communication state with chamber 6 by pipeline 15.Valve 16 control carbonic acid gas and antigen circulation are through piping 15.The heating tool (not shown) is below chamber 6.A rotatable blade 17 is arranged in chamber 6, can operate antibody and blood are mixed in chamber 6.When antibody and blood remain in the chamber 6, timing tool (not shown) monitoring incubation time.Hatch and may be displayed on remaining time on the control panel indicating meter 18.

The output storage vessel is promptly exported blood bag 19 and is also hung in the equipment 1, and interconnected by pipeline 20 and chamber 6.Pipeline 20 is through heat sealers 21, and it can be through operation and sealing-duct 20, and sealing output blood bag 19 thus.Another peristaltic pump (not shown) is provided, has been used for the content of chamber 6 is pumped into output blood bag 19.

Chamber 6 and pipeline 7,20 and output blood bag 19 are disposable article, can abandon after each program.

In operation, the user will export blood bag 19, chamber 6 and pipeline 7,20 insertion equipment 1.Pipeline 20 is through heat sealer 21 and peristaltic pump.Pipeline 7 is through another peristaltic pump and Coulter-counter flow cell 9.The user also inserts the blood bag 4 that contains patient's blood sample and it is attached to pipeline 7.Except above-mentioned, the user is attached to aseptic filter membrane 13 with the pipeline 15 of chamber 6.The user closes the front panel 3 of shell 2 then, and with the keyboard starting outfit 1 of this equipment.

Equipment 1 automatic weighing input blood bag 4.The weight of input blood bag 4 sends to the central computer system (not shown) of the equipment of being arranged in 1 automatically.According to the weight of input blood bag 4, computing system can be determined the blood volume of equipment 1.Peristaltic pump is pulled out through piping 7 blood from input blood bag 4 then.Blood flow is through the transparent window of pipeline 7, and Coulter-counter 9 determines to pass through the cell concn of pipeline 7.Coulter-counter directly is sent to central computer system with signal.Central computer system is determined from the blood volume calculated and cell concn need be by the one or more correct volumes that are injected to the antibody in the chamber 6 the syringe 10,11.Peristaltic pump continues to pump blood from blood bag 4, and up to receiving signal from central computer system, this signal stops pump.The signal reaction that sends from central computer system is in the signal that sends to central computer system from Coulter-counter 9, feels when Coulter-counter 9 and sends this signal when not having cell again through window 8.

Central computer system is sent signal to the stepper-motor (not shown) that is attached to

syringe

10,11 then, makes the one or more antibody of sending volume calculated by pipeline 15 in chamber 6 in the

syringe

10,11.

Selectivity is introduced carbonic acid gas (switching mechanism of valve 16 can be controlled by central computer system) by opening valve 16 from carbonic acid gas inlet 14 then.The amount of carbon dioxide that adds is calculated according to the volume in known chamber 6, pipeline 7,20 and the output blood bag 19 and from the blood volume that input blood bag calculates by central computer system.Carbonic acid gas final concentration in the chamber 6 is controlled to be about 5%.By pipeline 15 carbonic acid gas is introduced chamber 6.Then carbonic acid gas is blown over any residue antibody in the pipeline 15, guarantee that the antibody of all releases is added into chamber 6.In case 4 emptyings of input blood bag just become air bag, are used to hold carbonic acid gas and introduce the extra gas volume in back.

This equipment can further comprise a well heater (not shown) under central computer system control, and this well heater is positioned at below the chamber 6, and the temperature of chamber 6 is controlled at 25-37 ℃.The thermostatted that is connected with well heater prevents superheated or underheating.

Then blood and antibody reagent are hatched one section preset time in chamber.Suitably, incubation time is no more than 24 hours, preferred 4-8 hour.The time of selecting should enough allow to take place reverse differentiation.

Between incubation period, vane-type arm 17 slowly rotates, so that mixed antibody and blood.

When finish hatch after, vane-type arm 17 continues rotation, and as the curette arm, removes cell that is attached to chamber 6 surfaces and the results that therefore promote cell.Peristaltic pump is pulled out the content (promptly containing undifferentiated cell, i.e. the blood of stem cell) of chamber 6 from the bottom of chamber 6, make it enter output blood bag 19.This pump continues to pump, and has entered output blood bag 19 up to the blood of measurement volumes (determining by the peristaltic pump that uses calibration).

At last, heat sealer 21 is clamped pipeline 20, makes the length of one section about 2cm of pipeline 20 sealed, and sealer 21 cuts off pipeline 20 in about mid-way of sealing place then.

Equipment 1 can remind the user that this process is finished then.

The user can take out output blood bag 19 then.Can discard chamber 6, pipeline 7,20 and input blood bag 4.

If desired, output blood bag 19 can be transferred in the purification system, for example is used to identify the system of the cell of the characteristic cell surface marker with undifferentiated cell, can be used as indication although profile changes also.Can remove antibody reagent and/or also can choose enrichment wantonly and not break up (doing) cell with purification system.Suitable purification system is the CliniMACs/Isolex system.

II. undifferentiated cell and noble cells

By many undifferentiated cells and noble cells, there are many common instructions about them this area in the body.

About hematopoietic cell be, can reference example as, but be not limited to Levitt andMertelsman 1995 (Haematopoietic Stem Cells, published byMarcel Dekker Inc-especially pages 45-59) and Roitt et al. (Immunology, 4th Edition, Eds.Roitt, Brostoff and Male 1996, Publ.Mosby-especially Chapter 10).

Undifferentiated cell is a kind of immature cell, and it does not show sophisticated differentiating characteristic, but can produce the offspring of the sophisticated differentiating characteristic of performance.A well-known example of undifferentiated cell is a stem cell.

Stem cell is a kind of undifferentiated immature cell, can self (unrestricted division) and differentiation (specialization).Developmental embryo has many immature cells; Yet their number constantly reduces in growth course.On the contrary, the adult biology contains the stem cell of limited quantity, is confined in some body cavity.

It is generally acknowledged that stem cell is monoenergetic, dual intensity or polyenergic.

Monoenergetic and dual intensity stem cell are more restricted in growth, and produce the specific cell of one or both types respectively.On the contrary, multipotential stem cell (PSCs) can be divided into many dissimilar cells, produces tissue (constituting organ by it) or under the situation of myeloid-lymphoid stem cell, forms whole organism.

Multipotential stem cell is different with monoenergetic or dual intensity stem cell, and it can carry out the polyphyly differentiation, produces the tissue of being made up of the cell aggregation of dissimilar or clone.

Hemopoietic stem cell is an example that is present in the multipotential stem cell in the myelocyte, and it can produce various hemocytes (comprising white corpuscle and red corpuscle).

Blood is a kind of tissue juice, contains lymphocyte (Ly), monocyte (Mo), neutrophil leucocyte (Ne), basophilic granulocyte (Ba), eosinophilic granulocyte (Eso), thrombocyte (PI) and red corpuscle (Rbc), sees Fig. 3.The tissue of specialization is produced by the differentiation of hemopoietic stem cell (Hsc).Generally speaking, (in systemic circulation) infected in the white corpuscle opposing, and red corpuscle (in partial circulating) transports nutrition, oxygen and refuse in health.

In the past, hemopoietic stem cell by from (i) marrow, (ii) transforming growth factor peripheral blood or (iii) the Cord blood (placenta) separation extract hemopoietic stem cell.Recently, from embryo's hemopoietic stem cell of having done (ES) cell preparation, they extract from the embryo with technology in vitro fertilization.These undifferentiated cells can polyphyly differentiation and rebuild all bodily tissues, that is, be polyenergic.

Extracting method above-mentioned is a trouble, is dangerous sometimes, and in question in some cases for not meeting ethics, particularly in the embryonic stem cell extracting method.

The undifferentiated stem cell that has many hematopoietic cells to be.These comprise multipotential stem cell (PSCs), lymphoid stem cells (LSCs) and myeloid stem cell (MSCs), concentrate to be called lymph hemopoietic progenitor cell (LPCs).

LSCs and MSCs are being differentiated to form by PSCs.Therefore, LSCs and MSCs more finalize the design than PSCs.

The undifferentiated cell of hematopoiesis system comprises T cell, B cell, eosinophilic granulocyte, basophilic granulocyte, neutrophil leucocyte, megalokaryocyte, monocyte, red corpuscle, granulocyte, mastocyte and lymphocyte;

T cell and B cell being differentiated to form by LSCs.So T cell and B cell are more finalized the design than LSCs.More specifically, the differentiation chain is LSC → pro B lymphocyte (pro-B-cell) or prothymocyte.Pro B lymphocyte → pre B cell (pre-B-cell) → mature B cell → plasmocyte.Prothymocyte → common thymocyte → ripe thymocyte (assist/inducing or cell toxicant/inhibition clone)-see Fig. 4.

Eosinophilic granulocyte, basophilic granulocyte, neutrophil leucocyte, megalokaryocyte, monocyte, red corpuscle, granulocyte, mastocyte, NK cell and lymphocyte being differentiated to form by MSCs.So each in these cells is all more finalized the design than MSCs.More particularly, the differentiation chain is MSC → prematurity megakaryoblast (→ megakaryoblast → megalokaryocyte → thrombocyte) or primitive erythroblast (→ protoerythrocyte → skein cell → red corpuscle) or myelocyte monocyte stem cell, promptly is divided into the dual intensity stem cell of myeloblast (→ promyelocyte → myelocyte → granulocyte) or monoblast (→ promonocyte → monocyte → huge have a liking for cell)-see Fig. 5.

The extensively firm differentiation pathway of hemopoietic is according to the special cell surface marker of profile and clone identification various cell stages (as follows) easily.

Other stem cells comprise the neural stem cell that can produce neurone, stellate cell and few prominent cell, multipotential stem cell (Nakafuku and Nakamura, 1995, J.Neurosci Res., vol 41 (2): 153-68; Anderson, 1994, FASEB J., vol 8 (10): 707-13; Morshead et al., 1994, Neuron, Vol 13 (5): 1071-82).The skeletal muscle satellite cell is the stem cell of another kind of type, and a kind of unique types of the myogenous cells of more specifically saying so can remain the immobilized stem cell in the adult, and can produce new muscle cell (Bischoff when needed, 1986, Dev Biol., vol 115 (1): 129-39).The stem cell of other types is a kind of hypotype, endothelial stem cell and mescenchymal stem cells of epithelial stem cell, basilar cell.

A kind of very important stem cell type is embryo (ES) stem cell.These cells are widely studied and identify.In fact, the ES cell is used to produce transgenic animal by routine.Proved the ES cell in vitro differentiation for some cell types, comprise lymphocyte precursor (Potocnik et al., 1994, EMBO J., vol 13 (22): 5274-83) and neurocyte.US 5,843,780 and US 6,200,806 separation of primate es cell is disclosed.The ES cell is characterised in that the mark of many phasic specificities, as phasic specificity embryo mark 3 and 4 (SSEA-3 and SSEA-4), high molecular glycoprotein TRA-1-60 and TRA-1-81 and alkaline phosphatase (Andrews et al., 1984, Hybridoma, vol 3:347-361; Kannagi et a/.1983, EMBO J., vol 2:23n 361; Foxet al., 1984, Dev.Biol., vol 103:263-266; Ozawa et al., 1985, Cell.Differ., vol 16:169-173).

Many antigens are relevant with undifferentiated cell and noble cells." be correlated with " and represent that herein cell expressing maybe can express or present or can be induced to presenting or comprising separately antigen.

Most of undifferentiated cells and noble cells comprise main histocompatibility (MHC) I class antigen and/or II class antigen.If these antigens are relevant with these cells, they are known as the I class so ⁺And/or II class ⁺Cell.

Every kind of specific antigens relevant with undifferentiated cell or noble cells can be used as a kind of mark.Therefore, dissimilar cell can be distinguished mutually according to its relevant specific antigen or according to the particular combinations of related antigen.

These labelled antigen examples comprise antigens c D34, CD19 and CD3.If there is these antigen, these specific cells are known as CD34 respectively so ⁺, CD19 ⁺And CD3 ⁺Cell.If there is no these antigens, these cells are known as CD34 respectively so ^-, CD19 ^-And CD3 ^-Cell.

More specifically, PSCs is CD34 ⁺DR ^-TdT ^-(other useful marks are CD38 to cell ^-And CD36 ⁺).LSCs is DR ⁺, CD34 ⁺And TdT ⁺Cell (also is CD38 ⁺).MSCs is CD34 ^-, DR ⁺, CD13 ⁺, CD33 ⁺, CD7 ⁺And TdT ⁺Cell.The B cell is CD19 ⁺, CD21 ⁺, CD22 ⁺And DR ^-Cell.The T cell is CD2 ⁺, CD3 ⁺And CD4 ⁺Or CD8 ⁺Cell.The prematurity lymphocyte is CD4 ⁺And CD8 ⁺Activated T cells is DR ⁺Cell.Natural killer cell (NKs) is CD56 ⁺And CD16 ⁺The T lymphocyte is CD7 ⁺Cell.Lymphocyte is CD45 ⁺Cell.Granulocyte is CD13 ⁺And CD33 ⁺Cell.Monocyte is huge, and to have a liking for cell be CD14 ^-And DR ⁺Cell.Other detailed content is seen Fig. 4 and Fig. 5.

Embryonic stem cell is expressed SSEA-3 and SSEA-4, high molecular glycoprotein TRA-1-60 and TRA-1-81 and alkaline phosphatase.They do not express SSEA-1 yet, and the existence of SSEA-1 is the indication of differentiation.The mark of the stem cell of known other types, for example Nestein of neuroepithelial stem cell (J.Neurosci, 1985, Vol 5:3310).Mescenchymal stem cell is SH2, SH3, CD29, CD44, CD71, CD90, CD106, male such as CD120a and CD124, and CD34, CD45 and CD14 feminine gender.

As selection or additional, many cells can be identified by resemblance.Can use microscope, and optional to use the staining technique identification of cell be a kind of branch that is greatly developed that is called histological science, person skilled has extensively been grasped this technology.Clearly cell dyeing will only carry out in the five equilibrium thing of cell, to confirm its feature, because dyeing can cause necrocytosis in general.

Therefore,, can identify some cell type (promptly this cell is noble cells or undifferentiated cell) by the existence of the antigenic mark enumerated more than seeking, and special cell type (promptly this cell is T cell or B cell).

Undifferentiated cell can comprise any with antigen presentation, catch or discern relevant any composition.Preferably, undifferentiated cell is a MHC I class ⁺And/or MHC II class ⁺Cell.

More the committed cell can comprise any with antigen presentation, catch or discern relevant any composition.Preferably, more the committed cell is a MHC I class ⁺And/or MHC II class ⁺Cell.

More the committed cell derives from any cell that undifferentiated cell maybe can derive from undifferentiated cell.For example, more the committed cell can be lymphoid stem cells or myeloid stem cell, and undifferentiated cell is a multipotential stem cell.

In another kind of preferred embodiment, more the committed cell is a noble cells, for example the CFC-T cell, the CFC-B cell, CFC-Eosin cell, CFC-Bas cell, the CFC-Bas cell, CFC-GM cell, CFC-MEG cell, the BFC-E cell, the CFC-E cell, T cell, B cell, eosinophilic granulocyte, basophilic granulocyte, neutrophil leucocyte, monocyte, megalokaryocyte, red corpuscle, undifferentiated cell are myeloid stem cell, lymphoid stem cells or multipotential stem cell.

If more Ding Xing stem cell is a kind of noble cells, so noble cells preferably bone-marrow-derived lymphocyte (activated or unactivated), T lymphocyte (activated or unactivated), hugely have a liking for the monocytic cell of cell, can express I class or the antigenic karyocyte of II class, can be by abduction delivering I class or antigenic cell of II class or cytode (promptly not containing nucleolate cell, for example red corpuscle).

In another kind of preferred embodiment, noble cells is selected from large granular lymphocyte, null lymphocyte and natural killer cell, and each all expresses CD56 and/or CD16 cell surface receptor.

Noble cells even can produce by the karyocyte stoning.

I11. reagent

The reagent operability relates to the more cell of differentiation, so that make the reverse noble cells that is divided into of this cell.In this, being used to make more, the reverse reagent that is divided into undifferentiated cell of committed cell can directly or indirectly relate to more committed cell.

This reagent can play in the cell in committed cell more and acts on.Yet this reagent preferably works in the extracellular of more finalizing the design.

The surface that an example that directly relates to is more committed cell has at least a cell surface receptor, as has a cell of the β chain (usually find with same or similar sequence zone) of homologous region, homologous region on described homologous region such as the B cell, wherein this reagent directly relates to cell surface receptor.Another example is that more committed cell's surface has cell surface receptor, as has a cell of the α chain (usually find with same or similar sequence zone) of homologous region, homologous region on described homologous region such as the T cell, wherein this reagent directly relates to cell surface receptor.

Another example that relates to indirectly is that more committed cell's cell surface has at least two kinds of cell surface receptors, and this reagent relates to one of them can influence another kind of acceptor, and this induces more committed cell's reverse differentiation subsequently.

Making more, the reverse reagent that is divided into undifferentiated cell of committed cell can be compound or composition.Yet this reagent preferably can relate to the cell surface receptor of the cell surface of more finalizing the design.Like this, in a kind of preferred embodiment, this reagent operability relates to a kind of acceptor-this receptor that is present in the cell surface of more finalizing the design can be by the cell expressing of more finalizing the design, as the acceptor that can be expressed by the committed cell.

For example, preferred reagent comprise any one or multiple cyclic amp (cAMP), CD4 molecule, CD8 molecule, TXi Baoshouti partly or entirely, part (fixed or free), peptide, TXi Baoshouti (TCR), antibody, cross reacting antibody, monoclonal antibody or polyclonal antibody.Also can use somatomedin, as hemopoieticgrowth factor, erythropoietin and granulocyte-monocyte G CFS (GM-CSF) for example.

If this reagent is antibody, cross reacting antibody, monoclonal antibody or polyclonal antibody, this reagent antigenic β chain of MHC II class preferably so, the antigenic β chain of MHC HLA-DR, one or more in any one of the antigenic α chain of MHC I class or II class, the antigenic α chain of HLA-DR, MHC II class antigen or antigenic α of MHC I class and β chain or multiple antibody, cross reacting antibody, monoclonal antibody or the polyclonal antibody.Suitably an example of antibody is CR3/43 (being provided by Dako).

Noun " antibody " comprises maintenance in conjunction with active various fragments (being sheared or recombinant technology by proteolysis) and derivative, as Fab, and F (ab ') ₂With scFv antibody, with and stand-in or biological isomer.Antibody also comprises the genetically engineered variant, the some of them aminoacid sequence has passed through modification, for example increased combination by the substituted amino acid residue, wherein antibody results from the living species beyond the cell that need handle according to this clearly demarcated method, to reduce the possibility (example is " humanization " mouse monoclonal) of adverse immune response.

The cell transformation that is used to realize more to finalize the design is that the reagent of undifferentiated cell preferably works in the extracellular of more finalizing the design.Particularly, more the committed cell preferably comprises the acceptor that can be related to by this reagent operability, and this reagent operability relates to this receptor.

For example, this receptor can be a cell surface receptor.The specific examples of cell surface receptor comprises MHC I class and II receptoroid.Preferably, this receptor comprises α composition and/or β composition, as the situation of MHC I class and MHC II receptoroid.

More preferably, this receptor comprises the β chain with homologous region, for example has the homologous region of HLA-DR β chain at least.

More preferably, this receptor comprises the α chain with homologous region, for example has the homologous region of HLA-DR α chain at least.

This receptor is preferably the I class or the II class antigen of main histocompatibility complex (MHC).In preferred embodiments, cell surface receptor is the HLA-DR acceptor, DM acceptor, DP acceptor, DQ acceptor, HLA-A acceptor, HLA-B acceptor, HLA-C acceptor, HLA-E acceptor, HLA-F acceptor, or in the HLA-G acceptor one or more.In a more preferred embodiment, cell surface receptor is the HLA-DR acceptor.

This reagent is the antibody of acceptor preferably, and this reagent is more preferably monoclonal antibody.

The another kind of preferred example of this reagent is to regulate mhc gene to express, as MHC I class ⁺And/or MHC II class ⁺The reagent of expressing.

In a kind of preferred embodiment, this reagent and biological response modifier coupling.The example of biological response modifier comprises alkylating agent, immunomodulator, somatomedin, cytokine, cell surface receptor, hormone, nucleic acid, nucleotide sequence, antigen or peptide.Preferred alkylating agent is endoxan or comprises endoxan.

Other preferred biological response modifier comprises the compound that can raise MHC I class and/or MHC II class antigen presentation.In a kind of preferred embodiment, this can make with the reagent of MHC receptors bind and more effectively work.

Because any cell type all can be used for expressing MHC I class and/or MHC II class antigen, should provide a kind of method that is used to make the many kinds of reverse differentiation of cell, no matter their whether constitutive expression MHC I class and/or MHC II class antigen.

IV. make the method for the reverse differentiation of cell

In the method for the invention, the cell that makes a group comprise the committed cell contacts with the reagent that operability relates to one or more committed cells in the cell mass.Hatch this cell mass then,, and finally become undifferentiated cell so that reverse atomization takes place the reagent that allows to be related to by this reagent.

This contact procedure preferably comprises and relates to following one or more reagent: the homologous region of the homologous region of I class antigen α chain, II class antigen α chain, cd4 cell surface receptor, cd8 cell surface receptor, lymphocyte exist down homologous region, the lymphocyte of II class antigen β chain to exist down there are the homologous region of II class antigen α chain down in the homologous region or the lymphocyte of I class antigen α chain.Contact procedure is preferably carried out in the presence of biological response modifier (description of the face that sees before).

Usually, cell mass comes from biological sample, as blood or related tissue, comprises neuronal tissue, the muscle tissue of marrow, central nervous system or peripheral nervous system, the epidermis and/or the dermal tissue (for example oral cavity scraping blade) of skin.After preferably being born, biomaterial originates.Preferred whole blood or its processed products of using as blood plasma or buffy coat, can carry out under minimum medical supervision because take out them from the experimenter.General with antithrombotics such as heparin or Citrate trianion processing blood sample.Can handle the cell in the blood sample,, remove some cell type or some cell that from organize agglomerate, dissociates with some cell type of enrichment.The process useful that is used for purifying and isolated cell comprises that centrifugal (as density gradient centrifugation), flow cytometry and affinity chromatography (comprising the magnetic bead or the elutriation of the monoclonal antibody of cell surface marker as employing) (see VetteseDadey The Scientist (Sep.13 1999) 13 (18): 21).For example, Ficoll-Hypaque separates and can be used for removing red corpuscle and granulocyte, so that stay the cell with single nuclear, as lymphocyte and monocyte.

Because cell is primary culture substantially, must provide suitable nutrition to keep viability to cell mass.Suitable culture condition is well known to a person skilled in the art.Yet, pair cell group's processing preferably after from patient, taking out biological sample immediately, in general 12 hours, beginning in preferred 2-4 hour.Can use known technology, check cell viability as the blue exclusive method of platform.

Generally cell mass was hatched 2 hours at least with reagent, general 2-24 hour, preferred 2-12 hour, incubation temperature generally from about room temperature to about 22 ℃, about 37 ℃ of as many as comprises 33 ℃.Can from sample, take out a very little five equilibrium thing, check cell, thereby check the progress of reverse differentiation program with microscope and/or flow cytometry.Perhaps, this equipment can comprise trace tool, so that the progress of the reverse differentiation program of on-line monitoring.

In case the relative number of required cell type is increased to proper level, for example be low to moderate 0.1% or up to 5%, can use the cell mass of the change that obtains in every way.For the number of the undifferentiated cell that forms, importantly understand the multiplication capacity of stem cell.Although in some environment, the number of other undifferentiated cell of stem cell or formation can be very low, studies show that only 50% pluripotential hemopoietic stem cell just can be rebuild complete hemopoietic system in donor mice.So therepic use does not need to form a large amount of cells.

Also can carry out more the committed cell by the reagent that gives patient and pharmaceutical carrier or mixing diluents in the body to the conversion of undifferentiated cell.Yet reverse in many cases differentiation is external/stripped carrying out.

Can use treated cell mass subsequently, and only need processing seldom in external acquisition.For example, they can be to make up with a kind of pharmaceutically acceptable carrier or thinner simply, and need the patient of stem cell.

Yet, may need enrichment undifferentiated cell from cell mass, or from cell mass purifying cells.This can carry out easily by many methods (sees Vattese-Dadey-TheScientist 13 (18): 21 Sep 1999).Equipment of the present invention can be chosen wantonly and comprise purifying or separating tool, is used for the described undifferentiated cell of enrichment or reclaims described undifferentiated cell from the cell mass that changes.For example, can be with chromatography and/or flow cytometry according to cell surface marker and purifying cells.Yet the both unnecessary usually extensively purifying undifferentiated cell that also do not need from cell mass is because other cell that exists in the cell mass (for example stroma cell) may keep stem cell vigor and function.

Flow cytometry is a kind of establishment, reliable and strong technology that is used at mixed cellularity group identification of cell and sorting cells.Therefore, purifying or separating tool can comprise flow cytometer.Flow cytometry is to operate according to the particulate physical features in the liquid suspension, can distinguish these particles by the detection of a branch of light.These particles may be cell certainly.Physical features comprises cell size and structure, perhaps, and in recent years very generally with the monoclonal antibody bonded cell surface marker that is coupled to fluorescence molecule.

Kreisseg et al., 1994, J.Hematother 3 (4): mention among the 263-89, " owing to can obtain anti-CD34 monoclonal antibody; the flow cytometry of multiparameter becomes a kind of instrument that can select for use of judging hemopoietic stem cell and hemopoietic progenitor cell ", and continued to describe the ordinary skill that is used for quantitatively expressing the cell of CD34 with evaluation by flow cytometry.In addition, Korbling et al., 1994, Bone Marrow Transplant.13:649-54 has instructed according to the HLA-DR expression and by immunosorption and flow cytometry purifying CD34 thereafter ⁺Cell.As previously discussed, CD34 ⁺Be the useful mark relevant with stem/progenitor cells.

Flow cytometry technology according to other physical features sorting stem cell has also been arranged.For example, Visser et al., 1980, Blood Cells 6:391-407 has instructed can be according to size and structurizing degree separate stem cells.Grogan et al., 1980, BloodCells, 6:625-44 has also instructed, " can be from simple hemopoietic tissue with high with verifiable purity sorting survival stem cell ".

Except selecting the extracellular according to the existing of cell surface marker or other physical features (just selecting), also can be with negative standard enrichment, purifying cells group.For example, have clone specific marker such as CD4, CD8, the cell of CD42 and CD3 can take out from cell mass by flow cytometry or affinity chromatography.

A kind of very useful technology that is used for purifying cells relates to uses antibody or other affinity ligand that is connected with magnetic bead.With having cell surface marker, hatch magnetic bead as cell mass and the cell of CD34, CD34 catches affinity ligand.The sample tube that will contain cell places the magnetic sample thickener, and magnetic bead is attracted to the test tube two ends.Through behind the one or many washing step, interested cell by part or substantially fully from other cell purifying come out.In negative the selection, be not to wash and magnetic bead bonded cell, but liquid phase is kept by discarded liquid phase, will from cell mass, take out with magnetic bead bonded cell effectively then.

These purification process based on affinity ligand can be used for any cell type that maybe can identify the suitable mark on it of having identified.

Urbankova et al., 1996. (J.Chromatogr B Biomed Appl.687:449-52) have instructed by the gravity field flow fractionation and have separated micropreparation hemopoietic stem cell from mouse bone marrow cells suspension.Urbankova et al., 1996 further mention, and this method is used to identify stem cell from mouse bone marrow cells, because these cells are bigger than other cell in the marrow, can separate them from mixture.So the physical parameter beyond the cell surface marker can be used for purifying/stem cell enriched.

The cell mass that contains undifferentiated cell and purifying undifferentiated cell that produces by method of the present invention can remain on external with known technology.Usually, mammalian blood serum such as FBS have been added in employing, and optional limit growth medium such as the Hanks that adds autologous plasma, and RPMI 1640, DulbeccoShi minimum essential medium (DMEM) or IscoveShi improvement Dulbecco substratum are to provide the felicity condition of cell growth.In a kind of preferred embodiment, culturing stem cells on feeder layer such as stroma cell layer (see Deryugina et al., 1993, Crit Rev.Immunology, vol 13:115-150).Confirmed that the stroma cell secretion makes progenitor cell maintain the factor of undifferentiated state.The long-term cultivation system description of stem cell is in Dexter etal., 1977 (J.Cell Physiol, vol 91:335) and Dexter et al., 1979 (Acta.Haematol., vol 62:299).

For example, Lebkowski et al., 1992 (Transplantation 53 (5): 1011-9) instructed and can use based on adopting Covalent Immobilization in the technology purifying people CD34 of the monoclonal antibody of polystyrene surface ⁺Hematopoietic cell, the CD34 that obtains by this method ⁺Hematopoietic cell can maintain the survival rate greater than 85%.Lebkowski et al., 1993 (J.Hematother, 2 (3): 339-42) also instructed and how to classify and cultivator CD34 ⁺Cell.The summary of the whole bag of tricks is also seen Haylock et al., and 1994 (Immunomethods, vol 5 (3): 217-25).

Can use many external tests, measure the identity (also seeing embodiment) that confirms stem cell as CFC.Usually measure with long-term culture-initiating cell (LTC-IC) and detect very primary hemopoietic stem cell (Eaves et al.1991.J.Tiss.Cult.Meth.Vol 13:55-62).LTC-IC makes hematopoiesis keep 5-12 week.

The cell mass that comprises undifferentiated cell can be freezing with the purifying prepared product that comprises undifferentiated cell, so that use later on.Frozen cell and the proper technology that they are brought back to life are well known in the art.Equipment of the present invention can be chosen wantonly further to comprise and be used for the freezing freezing instrument that comprises the cell mass of undifferentiated cell and/or comprise the purifying prepared product of undifferentiated cell.

On the other hand, reverse differentiation preferably betides the cell of the buffy coat that comes from blood sample or the cell in the buffy coat." buffy coat " is illustrated in the leukocytic cream that forms between red corpuscle and the blood plasma, and when not solidified blood by blood plasma centrifugal or when allowing to leave standstill.

V. the ratio of normal differentiation cell among the transition cell group

In healthy tissues, various types of cells comprise that the differentiation and the relative number of undifferentiated cell are at constant normally sometime.For example, healthy 21 years old individual white blood cell count(WBC) generally is every liter about 8 * 10 ⁶Ml, wherein 27% is lymphocyte, the 6%th, monocyte, the 67%th, granulocyte.The level of these cells (comprise relative number and absolute cell counting) is interfered in disease, for example suffers from the situation among the patient of chronic B cell lymphocyte white corpuscle (B-CLL).

Whole blood or buffy coat can be laid on histopaque to remove granulocyte, thereby in the part of monocyte for example, disturb or the relative number (being ratio) of conversion noble cells, thereby realize that with the reverse undifferentiated cell that is divided into of noble cells wherein undifferentiated cell also is divided into various cell types to replenish the cell that is converted again with self (propagation).

After also can for example buffy coat (obtaining), removing (in the density gradient substratum centrifugal or bear selection with the magnetic bead of antibody sandwich) red corpuscle, thrombocyte, granulocyte, monocyte and T lymphocyte from the blood donors of health, interference or change the relative number (being ratio) of noble cells.This processing can be called the enrichment of negative selection or some differentiated cell types, and is an example of conversion tissue.When in IscoveShi improvement Dulbeccos substratum calcic substratum such as (ISDM), cultivating these cells, noble cells is with the reverse undifferentiated cell that is divided into, and undifferentiated cell is with self (propagation) and be divided into various cell types again to replenish the cell that is converted.

Find at first that in this process cell aggregation becomes colony (carrying out homotype cell aggregation), to carry out reverse differentiation.Cell transformation is a undifferentiated cell in case more finalize the design, and they have just obtained the ability of propagation, and develops into the various cell types of differentiation again, thereby replenishes the relative number that is converted cell.

The reverse differentiation instrument of normal differentiation cell proportion among the operability transition cell group, for example, antibody (pure and link coupled promptly is incorporated into fixing and free ligand, as magnetic, glass or polystyrene bead); Histopaque, LymphoPrep or be used for density gradient substratum according to the cell density isolated cell; Or dextran (for example can cause erythroprecipitin).Other suitable reverse differentiation instrument can identify that one piece of article in Vettese-Dadey (sees The Scientist (Sep.13 1999) 13 (18): 21).

With being converted cellular exposure in suitable sequestrant, comprise EDTA, EGTA and heparin (described sequestrant can be called biological response modifier) can cause even the reverse undifferentiated cell that is divided into of committed cell more.For example, will be converted cellular exposure in one section preset time of EDTA, culturing cell in containing the calcic substratum of cortisone can make and revise and decide the reverse undifferentiated cell that is divided into of type cell then.These cells then carry out self and participate in a kind of new differentiation pathway, produce the red corpuscle ancestors, as burst forming unit red corpuscle class (BFU-red corpuscle class).The red corpuscle ancestors can be cultivated and be expanded in long-time, and can be used for the treatment of erythrocyte disorder and red corpuscle shortage.

VI. change the dissociated ion concentration in the substratum that comprises cell mass

Noble cells is exposed to EGTA plasma sequestrant for some time, is containing the calcic substratum of hydrocortisone then, for example cultivate these cells among the IMDM, can cause the more reverse undifferentiated cell that is divided into of committed cell.These cells then carry out self and participate in a kind of new differentiation pathway, produce the megalokaryocyte ancestors, as colony forming unit-megalokaryocyte (CFU-Meg), and finally produce thrombocyte.

VII. the undifferentiated cell method of finalizing the design again

A kind of important application of undifferentiated cell of the present invention is at tissue reconstruction, the application during for example nervous tissue or hematopoietic cell are rebuild.This relates to the differentiation of the undifferentiated cell that is produced by method of the present invention.Its carrying out can be by only giving the patient undifferentiated cell, and allow the intravital natural physiological situation influence differentiation of patient, gives undifferentiated cell generally in specific interested site, as marrow, spinal cord or lung.The reconstruction that a specific examples of this method is a hemopoietic system or additional is for example at CD4 ⁺In the AIDS patient's of lymphopenia the situation.

In addition, can expand cell then, for example be used for the treatment of administration external realization differentiation (being also referred to as " typing again " herein).This generally undertakies by giving somatomedin, for example, be neuronal cell with the ES cytodifferentiation with vitamin A acid.Methylcellulose gum handle the back with marrow stromal cell and IL-7 cultivate altogether be used for the ES cytodifferentiation be lymphocyte precursor (Nisitani et al., 1994, Int.Immuno., vol 6 (6): 909916).Le Page (New Scientist 16 December 2000) has instructed the ES cell can be divided into pulmonary epithelial cells.Bischoff, 1986 (Dev.Biol., vol 115 (1): 129-39) instructed how the muscle satellite cell to be divided into ripe myofiber.Neural precursor can expand that (vol 41 (2): 153-168) for Nakafuku andNakamura.1995, J.Neurosci.Res. with alkaline fiber somatomedin and Urogastron.Hemopoietic stem cell can be used many somatomedins, comprise GM-CSF, erythropoietin, STEM CELL FACTOR and interleukin (IL-1, IL3 IL-6) expand---the summary of the various factors is seen Metcalf, 1989 (Nature, vol 339:27-30).

Potocnik et al., 1994 (EMBO J., vol 13 (22): 5274-83) even proved that it is hematopoietic cell that employing hypoxemia (5%) condition makes the ES cytodifferentiation.

Therefore, in a kind of preferred embodiment of the present invention, undifferentiated cell is fixed to the cell of typing again, as noble cells.Again Ding Xing cell can be the same cell system that produces the more committed cell of undifferentiated cell.In addition, again the cell of typing can with the more committed cell who produces undifferentiated cell homology not.For example, bone-marrow-derived lymphocyte can reversely be divided into CD34 ⁺CD38 ^-HLA-DR ^-Stem cell.Stem cell can be finalized the design again in B clone (same clone) or lymphocyte series (not homology) then.

Undifferentiated cell is fixed to the cell of typing again, and for example noble cells can be realized well known to a person skilled in the art variety of way.Known to the employing ad hoc fashion, and in defined medium, cultivate the cell that undifferentiated cell can realize being divided into selection.

Only illustrate, adopt following cultural method undifferentiated cell can be divided into the myocardial cell:

The 1st day: with normal density undifferentiated cell is transferred to the plate that scribbles gelatin, with the free fibroblastic culture that pollutes.

For normal transfer, use trypsin digestion and cell, leave up to colony.The soft processing is so that make cell keep loosely connected one-tenth agglomerate.With cell in no LIF ES cell culture medium directly to be laid on bacterium level Petri culture dish (as follows) at 1: 3.

The 3rd day: sucking-off substratum gently.Avoid the too much aggregation of sucking-off.Add new substratum then.

The 5th day: sucking-off substratum as the 3rd day, replace substratum.

The 7th day: cell is laid on 24 takes turns tissue culture level flat board.

The 9th day: the substratum and the observation that change half were beated.

The 11st day: the substratum and the observation that change half were beated.

Only be as an example, according to following program, undifferentiated cell can be divided into spongiocyte and neurone:

For normal transfer, use trypsin digestion and cell, leave up to colony.The soft processing is so that make cell keep loosely connected one-tenth agglomerate.Cell directly was laid on bacterium level Petri culture dish (obtaining from Sigma) with 1: 3 in containing the no LIF ES cell culture medium of 1 μ M all-trans retinoic acid.

The 3rd day: the collecting cell aggregation also was laid on tissue culture ware (about 25 the cell aggregation things of the tissue culture ware of each 6cm) again in the ES of no LIF or RA cell culture medium.Sucking-off substratum gently.

The 8th day: the substratum that changes half.From this day, at least 10% cell shows the neurone phenotype.They are by the cresol purple specific stain, and are strong positive to N-CAM antigen.

Those skilled in the art should understand the appropriate procedure that realization is fixed to undifferentiated cell any selected noble cells easily.

Undifferentiated stem cell of the present invention can be done (ES) cell culture technology and cultivate with the embryo of any routine.Only illustrate, the details of appropriate culture medium that is used for undifferentiated cell or ES cell cultures is as follows:

Be preparation 100ml substratum

DMEM(GIBCO cat#11965-062)	80ml
DMEM(GIBCO cat#11965-062)	80ml	15％FCS	15ml
Pen/Strep	1ml	15％FCS	15ml
Pen/Strep	1ml	L-glutaminate	1ml
MEM non-essential amino acid (GIBCO cat#11140-050)	1ml	L-glutaminate	1ml
MEM non-essential amino acid (GIBCO cat#11140-050)	1ml	Lif(10 ⁵U/ml)	1ml
BME(0.1M)	0.2ml	Lif(10 ⁵U/ml)	1ml

Lif is positioned in the ampoule of 1ml, as 10 ⁷The LIF ESGRO AMRAD of U/ml, it can dilute in 100ml DMEM and 10%FCS, and the five equilibrium thing with 5ml stores under-20 ℃.

For BME, 0.1ml BME (14.4M) can be added 14.3ml PBS, with the acrodisc filtration of 0.2 μ m, and under-20 ℃, store maximum 1 month.

Another kind of suitable substratum can be a PMEF substratum for example, and this substratum of 100ml can be by following concrete preparation:

DMEM(GIBCO cat&num；11965-062	88ml
DMEM(GIBCO cat&num；11965-062	88ml	10％FCS	10ml
Pen/Strep	1ml	10％FCS	10ml
Pen/Strep	1ml	L-glutaminate	1ml
BME(0.1M)	0.2ml	L-glutaminate	1ml

Other appropriate culture medium that is used to cultivate the ES cell will be conspicuous to those skilled in the art.

VIII. be used to identify the mensuration of reverse differentiation agents

Except above-mentioned reagent, can identify other reagent with WO96/23870 or measuring method of the present invention.On the other hand, the present invention provides also that be used to identify can be with the reverse method that is divided into the material of undifferentiated cell of committed cell/noble cells, this method comprises makes the cell mass that comprises the committed cell contact with candidate substances, and judge whether the relative number of undifferentiated cell increases in the described cell mass, and wherein said contact takes place in the presence of buffy coat.

Suitable candidate substances comprises and cell surface receptor bonded part, as antibody product (for example, mono-clonal and monoclonal antibody, single-chain antibody, the antibody that chimeric antibody and CDR transplant), for example with cell surface receptor bonded antibody.Interested especially cell surface antibody is described in front, comprises the MHC acceptor and has the CD title, as the surface protein of CD4 and CD8.Other and cell surface receptor bonded part comprise somatomedin.

In addition, can screen combinatorial library, peptide and peptide mimics, definite chemical entities, oligonucleotide and natural product libraries activity as reverse differentiation agents.Candidate substances can be used for initial screening in batches, and for example, 10 kinds of materials of each reaction screening are checked the material that shows inhibition in screening in batches individually.

Typical mensuration comprises that the branch things such as cell that will comprise the committed cell are positioned over proper container, in porous plate.Candidate substances is added in the hand-hole, cell is hatched in the hole.Hatch generally and approximately carrying out under the room temperature, or, for example about 22 ℃ to 37 ℃, comprise 33 ℃ from about room temperature.

Reverse differentiation can be by taking out a little five equilibrium thing of cell, and judge by microscope and/or flow cytometry whether the undifferentiated cell number changes and measure.Usually, the mensuration that the undifferentiated cell number changes is to change by the cell number that monitoring has a characteristic cell surface marker of undifferentiated cell to carry out, and also can be used as reference although profile changes.Suitably the example of cell surface marker comprises CD34 ⁺As selecting or replenishing, also can monitor typical cells surface markers with noble cells, and do not have the reduced number of cell of the typical cells surface markers of undifferentiated cell, for example monitoring has clone specific marker such as CD3, the relative reduced number of the cell of CD4 and CD8.

Preferably, the cell count increase with characteristic feature of undifferentiated cell occurred in 24 hours, and in preferred 4-8 hour, so these changes can not be explained by cell proliferation fully.

May need prescreen and for example cell surface receptor, as MHC I class or MHC II receptoroid bonded reagent.Any being accredited as with target cell surface receptor bonded reagent can be used for said determination then, to judge their effects to reverse differentiation.As special case, can with expressing antibodies in conjunction with the phage display library in territory identify with the target cell surface markers as the homologous region bonded antibody fragment of MHC II receptoroid β chain (be scFvs).Suitable is well known in the art in conjunction with measuring, as the generation and the screening of phage display library.These are measured and also can be used to identify optimized antibody or antibody fragment, and for example screening has shown the mutagenesis library of the antibody derivatives of realizing reverse differentiation.

IX. purposes

The invention provides the reverse method and apparatus that is divided into undifferentiated cell with the committed cell.Particularly, the invention provides from noble cells more and prepare the method and apparatus of stem cell.Its clinical meaning is huge, because stem cell is being used for the wide range of therapeutic purposes, but still very difficult up to now, trouble, and its acquisition can be subjected to ethical opposition sometimes.

The stem cell that produces according to the present invention can be used for recovering the specific cells group the patient, as hematopoietic cell group or its subgroup, as CD4 T lymphocyte.The more committed cell who is used to produce stem cell can come from the donor of same patient or coupling.Therefore the stem cell that produces according to the present invention can be used for curing and rebuilding specific cell tissue and organ.For example, undifferentiated cell can be used to produce the cell of committed cell as the lining alveolar, has therefore produced to substitute or to repair to destroy or the mechanism of diseased lung tissue (see Le Page, New Scientist 19 December 2000, p 20).

Therefore, the stem cell that produces according to the present invention can be introduced the patient, thereby recovers cell in vivo.Suitably, at first transplant stem cell, recover then.

Therefore, the present invention also comprises a kind of medicine, and this medicine comprises the undifferentiated cell by any one or this equipment preparation in these methods with suitably thinner, carrier or mixed with excipients.

In one embodiment, the medicine that comprises undifferentiated cell can be used to produce useful more committed cell, as the cell with correct gene group structure, and the more committed cell with wrong genome structure causes or relevant symptom or situation so that alleviate.Therefore, the present invention also provides the method for the sudden change that a kind of removal obtains from committed cell more, this method comprises that the method according to this invention forms a kind of undifferentiated cell, with not a kind of cell of typing again of undifferentiated cell typing, wherein the arrangement of the genome of this cell and/or nuclear or rearrangement cause this sudden change to be removed.

This gene preferably is inserted into genomic immunoglobulin domain or TCR district.

Disease or disorderly method that the present invention also provides a kind of treatment to suffer from by deficient cells or do not need cell to cause, this method comprises that the reverse reagent that is divided into undifferentiated cell of committed cell prepares undifferentiated cell by making the cells contacting of more finalizing the design cause more, the optional then cell of finalizing the design again that undifferentiated cell is fixed to, undifferentiated cell wherein, again the typing the impact cell deficient cells or do not need cell, thereby alleviate this disease or disorderly symptom, or cure the patient who suffers from this disease or situation.

In addition, can produce more committed cell with undifferentiated cell, this cell produces a kind of material object, and it can be cured, and any more committed cell with wrong genome structure causes or relevant symptom or situation.

For example, the present invention can be used to prepare antigenic antibody or the TXi Baoshouti that is divided into the cell expressing of more finalizing the design of undifferentiated cell by reverse.In this, antigen can be fetus specific antigens or cross reactivity fetus specific antigens.

The present invention also comprises the control undifferentiated cell and the method and apparatus of committed cell's level more.For example, the present invention includes a kind of method, this method comprises that the method according to this invention forms a kind of undifferentiated cell, activates a kind of apoptogene then, thereby influences undifferentiated cell, for example makes its death.

In a kind of preferred embodiment, the present invention relates to a kind of gene be introduced the genomic method of undifferentiated cell, wherein this method comprises gene is introduced more committed cell, and the method according to this invention prepares undifferentiated cell then, and this gene appears in the undifferentiated cell thus.

In a kind of preferred embodiment, the present invention relates to a kind of gene be introduced the genomic method of undifferentiated cell, wherein this method comprises gene is inserted in committed cell's more the genome, and the method according to this invention prepares undifferentiated cell then, and this gene appears in the undifferentiated cell thus.

This gene can be a kind of make undifferentiated cell and more noble cells obtain antipathogen more from it and infect gene as virus infection.Particularly, for example, can produce stem cell, through through engineering approaches its anti-AIDS be infected then with AIDS patient's bone-marrow-derived lymphocyte.When expanding and introduce among the patient, the helper T lymphocyte that obtains also can resist HIV to infect.

In another embodiment, the present invention relates to a kind of method of gene being introduced undifferentiated cell, wherein this method comprises gene is inserted in committed cell's more the genome, and the method according to this invention prepares undifferentiated cell then, and this gene appears in the undifferentiated cell thus.

In addition, the present invention also comprises the method that is used to prepare undifferentiated cell among the present invention, and wherein this method comprises the cell that undifferentiated cell is fixed to again typing, the cell that will finalize the design again and myelomatosis fusion then.The product that this allows vivoexpression to need in a large number, for example antibody or antigen or hormone etc.

The present invention includes undifferentiated cell with any one preparation in the method for the present invention.

In another embodiment, equipment of the present invention and/or method can be used for effectively cultivating the red corpuscle ancestors, are used to produce red corpuscle, and these red corpuscle can be used for for example shortage of supplemental blood supply.Suitably, method of the present invention can be used to produce megalokaryocyte, for use in producing thrombocyte.

In another embodiment, equipment of the present invention and/or method can be used to prepare the undifferentiated cell storehouse, promptly treated cell bank.

Others of the present invention comprise:

Any reagent of the present invention is in the purposes from the cell preparation undifferentiated cell of more finalizing the design.

The undifferentiated cell that the method according to this invention produces is in the purposes of following each side: produce monoclonal antibody or polyclonal antibody or the specific antibody any one from bone-marrow-derived lymphocyte or T lymphocyte; Produce the monocytic cell of scavenger cell; Generation can be expressed I class or the antigenic karyocyte of II class; Generation can be by abduction delivering I class or the antigenic cell of II class; Produce enucleate cell; Produce broken cells; Or generation apoptotic cell.

Otherwise the undifferentiated cell that the method according to this invention produces produce from bone-marrow-derived lymphocyte effector T cell or purposes.

The purposes of the undifferentiated cell that the method according to this invention produces in producing following any one or multiple material: medicine, as comprise bone-marrow-derived lymphocyte, the lymphocytic medicine of T or especially manufacturing the monocytic cell of medicine, scavenger cell, can express I class or the antigenic karyocyte of II class; Can be by abduction delivering I class or antigenic cell of II class or enucleate cell.

The present invention also comprises the method for utilizing above-mentioned purposes, and by the product or the composition of these methods preparation.

The present invention also comprises a kind of medicine, this medicine comprise undifferentiated cell of the present invention or by its acquisition, with the suitable product of thinner, carrier or mixed with excipients.

In a kind of preferred embodiment, this medicine comprises the antibody or the antigen from undifferentiated cell acquisition of the present invention of a kind of and suitable thinner, carrier or mixed with excipients.

This medicine is preferred for following any one disease of treatment: treatment or congenital metabolic trouble that cancer, autoimmune disease, hemopathy, cell or tissue degeneration, organ collpase, organ or tissue transplant.

Method of the present invention and the product that is obtained by these methods as undifferentiated cell, can be used for for example reverse differentiation of research, differentiation, and identify and study antigen newly developed and bunch differentiation antigen.

X. administration

The stem cell of the present invention and the cell of typing again, and show as can reverse noble cells reagent, can be used for the treatment of method.Cell of the present invention or reagent preferably make up with various compositions, to produce composition of the present invention.Said composition preferably makes up with pharmaceutically acceptable carrier or thinner, to produce pharmaceutical composition (can be used for the human or animal).Appropriate carriers and thinner comprise etc. and to ooze salts solution, for example phosphoric acid buffer.Composition of the present invention can be used for direct injection.Said composition can be through preparationization, is used for parenteral, intramuscular, intravenously, subcutaneous, intraocular, oral or percutaneous dosing.

The composition that comprises cell is generally by injection or inhibition administration.Can or be embedded in supported matrix in suspension, as sending in natural and/or the synthetic biodegradable matrix.Natural substrates comprises collagen stroma.Synthetic biodegradable matrix comprises polyanhydride and poly(lactic acid).These matrix are preferred for non-hematopoietic cell for external loose cell provides support.

Sending also can be sending of control, and that promptly controls in the time from several minutes to several hours or several days sends.Send and to be (for example by the intravenous injection) of whole body or directly to be delivered to specific purpose site.

Generally with 1 * 10 ⁵-1 * 10 ⁷The dosage of individual cell/kg gives cell.For example, for the patient of a 70kg, the construction haemal tissue of attaching most importance to can give 14 * 10 ⁶Individual CD34 ⁺Cell.

Route of administration described herein and dosage be intended to as just a kind of up to because those skilled in the art can judge route of administration and dosage for any patient and situation easily.

All following detailed method all are applicable to equipment of the present invention.

A. material and method

Patient

With the anticoagulant tube that contains EDTA from suffering from the leukocytic patient of B cell chronic lymphocytic, suffers from the antibody defective patient of (comprising the chain baby's Hypogammaglobulinemia of IgA defective and X), patient with HIV infection and acquired immune deficiency syndrome (AIDS), the patient who suffers from the He Jiejin lymphomas, suffers from the leukocytic patient of acute T cell, the 6 age in days babies that suffer from blastcytosis, patient with various infection and clinical condition, the Cord blood of healthy blood donor, the bone-marrow-derived lymphocyte preparation of marrow and enrichment obtains blood sample.

Clinical and laboratory condition

Patient's clinical and laboratory treatment condition comprises that the various treatment types of the blood sample that is applied to them all are described in table 1.Obtain differential white corpuscle (WBC) counting with Coulter-counter, these all are included in identical table 1.

The processing of blood

In case the acquisition sample is introduced its pure monoclonal antibody (DAKO) with the homologous region of HLA-DR antigen β chain in the chamber, and was at room temperature mixed maximum 24 hours.Some samples at first mixed 15 minutes, hatched in chamber under 22 ℃ then.The concentration that adds the monoclonal antibody of blood sample is 10-50 μ l/ml blood.

In addition, apply other processing with same concentration, comprise the monoclonal antibody of homologous region of monoclonal antibody, the PE link coupled HLA-DR antigen β chain of monoclonal antibody, the CD8 of monoclonal antibody, the CD4 of monoclonal antibody, the antigenic homologous region of I class of the homologous region that adds HLA-DR antigen α chain.

Other processing comprises the monoclonal antibody that adds the homologous region of HLA-DR antigen α chain and β chain simultaneously in blood sample simultaneously.

In addition, the pure monoclonal antibody combination with the homologous region of alkylating agent such as endoxan and HLA-DR antigen β chain adds in the blood sample.

After these are handled, with the monoclonal antibody of a group echo, blood sample is dyeed according to the explanation of manufacturers, use flow cytometry then.

With the incubation period of monoclonal antibody be 2 hours, 4 hours, 6 hours, 12 hours to 24 hours.

The antibody of mark

By flow cytometry, adopt the mark on the following cell of following monoclonal antibody detection: CD19 and CD3, CD4 and CD8, DR and CD3, CD56﹠amp; 16 and CD3, CD45 and CD14, CD8 and CD3, CD8 and CD28, simultest contrast (IgG1 FITC+IgG2a PE), CD34 and CD2, CD7 and CD 13﹠amp; 33, CD 10 and CD25, CD5 and CD 10, CD5 and CD21, CD7 and CD5, CD13 and CD20, CD23 and CD57 and CD25 and CD45RA (Becton﹠amp; Dickenson and DAKO).Other mark can comprise CD71 (red corpuscle mark), CD61 (megalokaryocyte mark), glycophorin A (red corpuscle mark), AC133 (stem cell labeling), CD38 (primordial stem cell mark), CD90 (stem cell labeling) and CD117 (multipotential stem cell mark).

Each patient's blood sample, no matter it is that handled or untreated, comprise the buffy coat sample, all adopt a plurality of above-mentioned traget antibody group in the equipment of the present invention to analyze, change so that explain the immunophenotype of following the different treatment type, these are to carry out in the branch things such as difference of identical blood sample.Untreated samples and other control sample are dyeed and analyze simultaneously.

Flow cytometry

Explanation dialogue cell sample according to manufacturers dyes and cracking.Carry out flow cytometry with simultest or PAINT A GATE software (BDIS) on FACScan@, this software comprises the negative control traceback.Obtained 1000-20000 incident, and be stored in the list mode file.

Profile

Analyze profile with microscope and Wright's staining.

Prepare stem cell from B-CLL enrichment or purifying (or normal) lymphocyte or from the buffy coat of blood sample

In following program, should use Aseptic technique:

(A) monocyte separates

(i) by going up at Histopaque, Lymphoprep or any LSM (sp.grav 1.077) with 400g centrifugal 30 minutes, from peripheral blood/buffy coat sample, obtain monocyte.

(ii) collecting monocytic cell is in the tapered tube of 50ml, with 30ml Han Keshi balanced salt solution (the no Ca that contains 2% heat activated foetal calf serum (FCS) and 2mM EDTA or 0.6% Citrate trianion ²⁺And Mg ²⁺, Sigma) washing, and with 400g centrifugal 10 minutes.

(iii) after the washing, count and estimate its viability with platform indigo plant and hematimeter pair cell.

If (iv) the B cell counting is higher than 70% (20 * 10 ⁹/ L WBC), directly carries out A (vi).

If (v) the B cell counting is lower than 70% (20 * 10 ⁹/ L, WBC).Bear selection with Macs microballon or FacsVantage purification technique, such as following C part description.

(vi) with the cell precipitation thing with 3 * 10 ⁶/ ml is resuspended in IMDM substratum (100 μ g/ml Streptomycin sulphate), and this substratum contains 10%FCS (heat-inactivated) and 10%HS (heat-inactivated), adopts the autologous plasma of 20%-50%.

(not differentiation) cell is done in preparation the monocyte part that obtains from B-CLL patient's peripheral blood sample

Separate (A) scheme according to above-mentioned monocyte, just work as white blood cell count(WBC) and surpass 20 * 10 ⁶/ ml when wherein the white corpuscle more than 50% is the B cell, should not bear selection.

Following program can be carried out in equipment of the present invention.

(B) handle cell with pure CR3/43 (Dako) monoclonal antibody

When A (after having obtained monocyte vi) and separating, carry out following steps:

(i) will import the blood bag (on hanging over tenaculum from top A (vi) obtaining),

(ii) adopt the chamber with 6 holes, operational outfit is (to add the 2ml cell suspending liquid to each hole of this porous culture plate or chamber from top A (vi) obtaining) from input blood bag.

(iii) operational outfit, handle the hole of proper amt, (pure monoclonal antibody Dako), and is not handled other hole (negative control) to CR3/43 to add 99 μ l/ml (or be 49.5 μ l/ml for the buffy coat sample) from the syringe that contains CR3/43mab to every hole.

(iv) under the environment of humidity at 50%CO ₂, 37 ℃ (or 33 ℃) hatch chamber.

(C) cell purification

The method of known many separation and purifying cells (is seen Vettese Dadey Scientist 13 (18): 21S ep 13 1999).

A kind of appropriate means is with MACS microballon (Miltenyi Biotec, best explanation according to manufacturers) the B cell to be born selection herein:

(i) partly obtain monocyte according to A.

(ii) sedimentation cell and with 300 μ l/ altogether 10 ⁸The middle volume re-suspended cell of individual cell is in HBSS (containing 2%FCS and 2mM EDTA or 0.6% Citrate trianion).

(iii) add 100 μ l/ altogether 10 ⁸The pure monoclonal antibody of the CD2 of individual cell (IgG1, DAKO).

(iv) in same cell suspending liquid, add 50 μ l/ altogether 10 ⁸The pure monoclonal antibody of the CD33 of individual cell (IgG1, DAKO).

(mixture was at room temperature hatched 10 minutes.

(vi) use HBSS (containing 2%FCS and 2mM EDTA) washed cell and with same damping fluid with 400 μ l/ altogether 10 ⁸The final concentration of individual cell is resuspended.At 400g centrifugal 10 minutes.Resuspended precipitation (the same) in HBSS.

(vii) add the microballon of the anti-mouse IgG1 of 100 μ l rabbits mark/altogether 10 ⁸The individual cell explanation of manufacturers (or according to).

(viii) complete cell mixing and hatched 15 minutes at 6-12 ℃ (refrigerator).

(ix) with HBSS (containing 2%FCS and 2mM EDTA) washed cell, at 400g centrifugal 10 minutes, with same damping fluid with 500 μ l/ altogether 10 ⁸The final concentration of individual cell is resuspended.

(x) in the magnetic field of MACS separator, concentrate MS ⁺/ RS ⁺Post.If the monocyte count height adopts two MS ⁺/ RS ⁺Post.

(xi) with 3ml HBSS (containing 2%FCS and 2mM EDTA) washing column.

(xii) make cell cross post, use 4 * 500 μ l HBSS (containing 2%FCS and 2mMEDTA) washing then.

(xiii) wash-out and collecting cell be in tapered tube, precipitates then and be resuspended in IMDM and place as A part (blood bag vi).

D) FACS Vantage purifying B cell:

(i) from B-CLL peripheral blood of patients sample, obtain monocyte, such as above-mentioned A part description.

The combination of (ii) using CD19-PE and CD20-FITC link coupled monoclonal antibody is to these cell dyeings, to identify the B cell.

(iii) according to CD19/CD20 fluorescence, with Beckton Dickenson FACSVantage and the argon laser sorting about 10 that takes place with 488nm ⁷Individual cell.

(iv) wash the cell of purifying, and allow in the couveuse of humidity, at 5%CO with the Han Keshi balanced salt solution that contains 50%FCS ₂, 37 ℃ of following recovery are spent the night.

(v) precipitation and re-suspended cell place as (the vi) blood bag of Miao Shuing, the CR3/43 processing of partly describing with above-mentioned B then of A part.

In white corpuscle, prepare stem cell

Can in equipment of the present invention, in white corpuscle, handle cell with pure CR3/43 (Dako) monoclonal antibody:

(i) with 30-200 * 10 ⁹The white blood cell count(WBC) of/L is selected patient's (bone-marrow-derived lymphocyte of 73-95%).

(ii) collect blood to there not being the containing in the heparin test tube of Citrate trianion, EDTA or sanitas, blood is placed the blood bag by venipuncture.The preferred blood of collecting that uses immediately suitably, uses in about 6 hours after collection.Yet storage/refrigerated blood also can use in the back that thaws in nitrogen.

(iii) will be positioned over the equipment of the present invention from the blood bag that contains whole blood that (ii) obtains.

(iv) carry out automatic differential white blood cell count(WBC) with Coulter-counter.Preferred selection white blood cell count(WBC) is 30-200 * 10 ⁶The patient of/ml (wherein 60-90% is the B cell).

(v) also can assess the viability of blood automatically, appropriate means comprises iodate third ingot and flow cytometry or platform indigo plant and hematimeter, uses the ammonium chloride solution splitting erythrocyte then.

(vi) add CR3/43 antibody in the chamber of this equipment in whole blood sample, final concentration is 1-3 μ l/10 ⁶Individual cell is (for example, if the WBC counting is 50 * 10 ⁹/ L, so every ml blood should add 50 μ l CR3/43 monoclonal antibodies, and mouse IgG concentration is 159 μ g/ml).

(vii) thoroughly mix blood and antibody,, keep the scheduled time eventually at the chamber of hatching under preferred 18-37 ℃, promptly be no less than 2 hours in room temperature with the blade in this apparatus cavity.

(viii), adopt the trace tool of this equipment, analyze hemocyte with flow cytometry, colony mensuration, long-term cultivation and/or PCT adding mAbs after 0 hour, 2 hours, 6 hours and 24 hours.

Attention:, should before analysis, thoroughly mix and take a sample with the little head of pipette in wide hole owing to the homotypic aggregation of B cell and because inducing in the test tube bottom of mAb CR3/43 formed adherent cell.

Trace obtains unified cell mass in analytic process, before CR3/43 handles with the blood sample five equilibrium extremely in a plurality of chambers.

Prepare to analyze the stem cell that produces by the B cell of cultivating with the processing of CR3/43 monoclonal antibody

Can assess for example per 2 hours, 7 hours, 24 hours, every day, per 7 days or longer time period (replenish to behind the cell weekly with the long-term cultivation base, carried out in every month) at many time points with the stem cell that method of the present invention produces.Analyze one or more processing and control wells at each time point, after this analyze remaining processing and control wells in the successive time period.

Can assess automatically by the trace tool of inserting in the equipment of the present invention.

(i) remove non-adhesion layer gently with the little transfer pipet in wide hole, repeat to draw, destroy cell mass, thereby obtain single-cell suspension liquid with the little transfer pipet in wide hole.

(ii) scrape off adhesion layer, repeat to draw, destroy cell mass, thereby obtain single-cell suspension liquid with the little transfer pipet in wide hole with the cell curette.

(iii) or, at first with HBSS flushing, thereby use the tryptic digestion adhesion layer, the trypsinase of adding 2ml 0.25% in every hole then, and under 37 ℃, hatching 10 minutes.

(iv) repeat to draw, destroy cell mass gently with the little transfer pipet in wide hole.

(v) hatch with 20% FCS after 10 minutes and reach final concentration, thus deactivation trypsinase.

(vi) can be with (ii) above-mentioned or ((promptly handling and untreated cell) culturing cell of every type that each time point v) obtains merges, and under 400g centrifugal 10 minutes.

(, should directly use (the vi) cell precipitation of Huo Deing (promptly handle and untreated cell) vii) for carrying out pcr analysis (seeing C).

(viii) for carrying out the colony determination and analysis, (ii) or (v) the cell precipitation of Huo Deing (promptly handling and untreated cell) is resuspended among the IMDM that contains 2% hot activation FCS with above-mentioned steps.Can take out little branch things such as cell, pair cell is counted, and assesses their viability with platform indigo plant and hematimeter.

(ix) analyze for carrying out FACs, (ii) or (v) the cell precipitation of Huo Deing (i.e. processing and untreated cell) is resuspended in the Han Keshi balanced salt solution (no calcium and magnesium) that contains 2% hot activation FCS and 5mM EDTA above-mentioned steps.

Stem cell is analyzed:

Can be in order to following method assessment stem cell.Equipment of the present invention can comprise and is used to carry out method discussed in more detail below or its a part of trace tool.

(A) immunophenotype

Be the immunophenotype analysis (use flow cytometry) of carrying out whole blood sample:

(i) immunostaining (according to the explanation of manufacturers), lysed erythrocyte and handle the after scouring cell in incubation period with mAb.Generally can use dissolving and the washing soln buied from Becton Dickinson.

(ii) should be with the labeling of monoclonal antibodies red corpuscle that directly is coupled to fluorescein isothiocyanate (FITC) or phycoerythrobilin (PE) (being positioned at whole blood, monocyte part, the negative B cell of selecting of MACS or the B-CLL that sub-elects).

A group echo that (iii) is used for the immunophenotype analysis can be as follows:

CD38 PE+CD45 FITC+CD34 PE-Cy5

CD19 PE+CD10 FITC+CD34 PE-Cy5

CD117 PE+CD3 FITC+CD34 PE-Cy5

CD33 PE+CD61 FITC+CD34 PE-Cy5

Glycophorin A PE+CD71 FITC+CD34 PE-Cy5

AC133 PE+CD90 FITC+CD34 PE-Cy5

CD19 PE+CDS FITC

IgG1 PE+IgGI FITC+IgG1 PE-Cy5 (isotype negative control)

(iv) can adopt the double-tagging of IMK+ test kit (Becton Dickinson): by following monoclonal antibody to forming:

CD45-FITC and CD14-PE;

CD19-PE and CD3-FITC;

CD8-PE and CD4-FITC;

HLA-DR-PE and CD3-FITC; And

CD56, CD16-PE and CD3-FITC.

The isotype coupling negative control that also comprises IgG1-FITC and IgGa-PE..

(following other antibody that v) also can use Dako and Becton Dickinson to produce:

The PE link coupled: anti-CD8, anti-CD 33 resists-13, anti-CD34, anti-CD19, anti-CD2, anti-CD14, anti-CD 33 and anti-CD5;

FITC link coupled: anti-CD3, anti-CD7, anti-IgM, anti-CD22, anti-CD20, anti-CD10, anti-CD7, anti-CD16, anti-TCR α β.

(vi) also can use following antibody

Also can use the IgG3 monoclonal antibody (Dako) of the special affinity purification of CD34, it can be with FITC or the anti-mouse immuning ball protein F of PE labelled goat (ab) ' ₂Fragment is as second antibody (DAKO).

Also use (PE-Cy5) the anti-CD34 of link coupled (Dako) of Quantum red (Quantum Red).

(vii) with FACScan or FACS Vantage (Becton Dickinson) or other flow cytometry analysis cell arbitrarily.Should analyze 100,000 cells and the writing time of equivalent.

(viii) use Proprietary Paint-a-Gate, Lysis II, Consort 30 and CellQuest software analysis data.

For carrying out the immunophenotype analysis (using flow cytometry) of buffy coat sample, for example

(ii) should be with single mark of the monoclonal antibody that directly is coupled to fluorescein isothiocyanate (FITC) or phycoerythrobilin (PE) or double-tagging red corpuscle.

Suitably, the mark that is labeled below can using:

CD34 (stem cell labeling); CD19 (bone-marrow-derived lymphocyte mark); CD45 (leukocyte marker); CD3 (T lymphocyte marker); CD33 (myelocyte); CD71 (red corpuscle mark); CD61 (megalokaryocyte mark); Glycophorin A (red corpuscle mark); AC133 (stem cell labeling); CD38 (primordial stem cell mark); CD90 (stem cell labeling); CD10 (lymphoid stem cells mark); CD117 (multipotential stem cell mark); IgG1 (negative control).

(B) profile:

For carrying out contour analysis:

Opticmicroscope

(i) with wide hole head of pipette cell thoroughly is resuspended among the IMDM that contains 2% heat activated FCS.

(ii) observe, adopt suitable staining fluid, for example adopt May and Greenwald staining fluid to analyze myelocyte and lymphocyte at the Leitz microscopically.Those skilled in the art readily understand the dyeing process that other is suitable for identifying other colony, for example Rui Shi or Giemsa staining.

(iii) can in blood smear or cell centrifugation prepared product, carry out the lymphocytic contour analysis of B-CLL respectively.

Laser Scanning Confocal Microscope

(i) obtain B cell (B-CLL or the healthy B cell that from the buffy coat of healthy blood donor, obtains) by foregoing

(ii) press foregoing CR3/43 monoclonal antibody treatments B cell.

(iii) in the organ culture ware, add 2ml cell suspending liquid (cover glass being housed) in this culture dish bottom.

The monoclonal antibody that (iv) adds 15 μ l CD19 FITC conjugates adds the monoclonal antibody of 15 μ l CD34PE/Cy 5 conjugates (Quantum is red).

(v) estimate viability, nuclear is dyeed with Hoechst with iodate third ingot.

(D) pcr analysis of VDJ/JHF gene rearrangement

VDJ and/or JHF district with PCR (Perkin Elmer thermal cycler) analysis IgH gene adopt before antibody treatment and the B-CLL peripheral blood of processing back (2 hours, 6 hours and 24 hours) and the template DNA that the buffy coat sample obtains.This scheme comes from Stolc et al (American Journal of Hematology 38:1-8; 1991).It is the positive detection of configuration immunoglobulin heavy chain gene that the JHF primer is used for planting.JH6 is as the positive internal contrast of immunoglobulin heavy chain gene.In leukemia and the normal lymphocytic B cell, the JHF district always is removed, and therefore can not be amplified, and internal contrast JH6 gene is kept perfectly.The β actin gene is with comparing.

(i) with Qiagen QiAmp Max blood test kit isolation of genomic DNA from whole blood.Adopt the maximum production scheme.Extract all DNA from each sample (processing with untreated).

(ii) on 1.5% sepharose, carry out the electrophoresis of pure dna and 1: 5 dilution DNA.

(iii) PCR-heated the pure genomic dna template of 2-3 μ L 5 minutes down at 96 ℃.

I)JH6a/JH6g JHFa/JHFg

" mastermix " that enough is used for 4 patients (handling and untreated 8 reactions) according to the detailed content preparation of following table.Can suitably amplify in proportion or dwindle.

	Volume/μ L	[end eventually]
	Volume/μ L	[end eventually]	The water PCR 10x damping fluid of nuclease free ^＊25mM MgCl ₂ ^＊5mM dNTPs 10μM P1 10μM P2 5U/μL Taq pol.	370 80 64 128 80 80 2	--1x 2mM 0.8mM 1 μ M 1 μ M 10 units

* each test tube of vortex 2-3 time before being used for the balance sample.

Contain the 95 μ L " mastermix " that add equivalent in the test tube of sex change genomic dna to each.With thermal cycler be set in 95 ℃ following 1 minute 30 seconds; 55 ℃ following 2 minutes; 72 ℃ following 1 minute 30 seconds (35 circulations); 72 ℃ following 5 minutes (1 circulation).

II) β Actin muscle

Preparation enough is used for I) described in 8 reactions) " mastermix ".Can suitably amplify in proportion or dwindle.

	Volume/μ L	[end eventually]
	Volume/μ L	[end eventually]	The water PCR 10x damping fluid of nuclease free ^＊25mM MgCl ₂ ^＊5mM dNTPs 10μM P1 10μM P2 5U/μL Taq pol.	380 80 48 128 80 80 2	--1x 1.5mM 0.8mM 1 μ M 1 μ M 10 units

* each test tube of vortex 2-3 time before being used for the balance sample.

The primer that is used for PCR

First group of segmental primer of 240bp that is designed for amplification IgH JHF gene is as follows:

JHFa AAA GGT GCT GGG GGT CCC CTG

JHFb CCC AGT GCT GGA AGT ATT CAG C

Second group of segmental primer of 242bp that is designed for amplification IgH JHF gene joining region 6 is as follows:

JH6a CAT TGT GAT TAC TAC TAC TAC TAC

JH6b GAT CCT CAA GGC ACC CCA GTG C

The 3rd group of segmental primer of 249bp that is designed for amplification β actin gene is as follows:

βACT-1 AAG GCC AAC CGC GAG AAG AT

βACT-2 TCG GTG AGG ATC TTC ATG AG

(D) DNA analysis of VDJ gene rearrangement

(i) come from processing with BamHI/HindII digestion with untreated peripheral blood sample or purifying B cell (coming from B-CLL patient)---generally need obtain the DNA of capacity with the cell in many holes to analyze.

(ii) Digestive system is dissolved in 0.8% the sepharose and and transfers to GeneScreen according to the explanation of manufacturers

Nylon membrane (Dupont) (Southern, 1975).

(iii) use ³²The P mark from the isolating people J of placenta genomic dna _HDna probe (Calbiochem, Oncogene Science) is analyzed the J district of IgH locus, thereby identifies the rearrangement of IgH gene.

(iv) self-developing film is placed-70 ℃ to descend several days, develop then.

(E) long-term cultivation:

Zhi Bei cell culture can be kept the longer time period (long-term cultivation) as stated above, (Iscove ' s improves Dulbeccos substratum (IMDM-Gibco BRL Life Technologies Ltd) to replenish the long-term cultivation base weekly, 10% heat activated FCS, 10% heat activated horse serum (HS), 1% hydrocortisone, 1% penicillin/streptomycin, 5 * 10 ^-7M stoste).

(i) at first, at certain time point, after 24 hours, handle, by in every hole, adding 2ml long-term cultivation base and diluting cells from CR3/43.

(ii) after the growth medium of removing half, in the hole, add substratum weekly.

(iii) use each hole of phase-contrast microscopy.

(F) colony is measured

(i) after each time period of handling beginning, can obtain the not adherent cell of 300 μ l in substratum by foregoing.

(StemCell Technologies is by the IMDM that contains methylcellulose gum, FCS (ii) to add 3ml methocultGFH4434 in the cell suspending liquid of above-mentioned substratum, BSA, L-glutaminate, rh STEM CELL FACTOR, rh GM-CSF, rh IL-3 and rh erythropoietin is formed).

(iii) take out 1.1ml cell mixture and bed board, spread 3 parts altogether.

(iv) with plate in the culture dish of humidity, 37 ℃ of following 5%CO ₂And 5%O ₂In hatched 14 days.

(v) check each hole before and after treatment in the CR3/43 monoclonal antibody with phase microscope.

(vi) can use flow cytometry, Laser Scanning Confocal Microscope and/or pcr analysis cell (colony) after 14 days.

(vii), should avoid ethanol gas colloidal sol,, hinder the survival (processing with untreated) of two types of cells because this can cause cell aggregation or destruction when carrying out colony when measuring.

B. result

CD19 and CD3 group

In equipment of the present invention, use the monoclonal antibody of the homologous region of HLA-DR antigen β chain to handle blood sample, always reduce CD19 ⁺The relative number of cell.This is labeled as full B cell antigen (seeing Table).This antigen is present on all human B lymphocytes in sophisticated all stages, but does not then have on the plasma cell of final differentiation.Therefore, this shows the reverse undifferentiated cell that is divided into of B cell.

Same processing causes CD3 ⁺The relative number of cell acutely increases, and particularly all the more so in the blood of B-CLL, this always follows CD3 ^-CD19 ^-The relative increase of cell number.CD3 is present on the thymocyte of all mature T lymphocytes and 65%-85%.Find this mark always with α-/β-or gamma/delta TXi Baoshouti (TCR) is related, these mixtures all are important for the signal transduction to cell interior.So this shows the reverse undifferentiated cell that is divided into of B cell, is fixed to new noble cells then again, i.e. the T cell.

A kind of new coexpression CD19 and the cell colony of CD3 mark appear in the B-CLL blood samples of patients of handling, i.e. CD19 ⁺And CD3 ⁺Cell (seeing chart 1, the

patient

2,3 and 4 of begin treatment after 2 hours, 6 hours or 24 hours), this processing can be carried out in equipment of the present invention.The relative number that other patient with different situations shows these cell colonies increases.These cells are very big, have a lot of particles, and express the CD19 of high level on their cytolemma.As if similar in the CD3 mark level of expressing on these cells to the CD3 mark level of on the normal mature lymphocyte, expressing.

In table 2, patient several 2,3 and 3 is reagent numerals of representing same patient, and they describe to be only used for representing in time the treatment effect to blood (laboratory and clinical condition for this patient see Table 1).

CD19 in treated sample ⁺CD3 ⁺As if colony reduced in time, reached the original level of measuring in the untreated samples at 2 hours, 6 hours and 24 hours.

Detect another kind of size and particle how many identical cell types in treated sample, these cell surfaces have high-caliber CD19 expresses, but does not have the CD3 mark, and is rich in the FC acceptor.As if yet the relative number of these cells reduces in time.Interesting is, after handling blood sample 24 hours (2,3 and 4), and the CD19 in one group of cell ^-CD3 ^-The relative reduced number of cell increases and observed this number after 2 hours and 6 hours at the processing blood sample.Yet the Ku Erte counting of the blood middle leukocytes group after the monoclonal antibody of the homologous region of usefulness HLA-DR antigen β chain is handled reduces.This processing of this discovery prompting has produced can not be by the cell (table 1) that is not true to type of Ku Erte count detection, but can be counted by measuring according to what flow cytometer of surface markers, size and particle.In addition, these cells that are not true to type can carry out contour analysis with Wright's staining and are counted at microscopically.The flow cytometry chart of these phenomenons is seen chart 1,2,3 and 4, the immunophenotype that obtains in blood sample is handled changes prompting CD19+ and the CD3+ lymphocyte is one group of cell that has a common boundary, but still keeps unique based on CD19 that compares with stem cell and CD3 relative expression.

In table 2, patient numbers 5 and 6 same patients of representative, but monitoring is in time handled and the analysis of the blood sample that is untreated respectively, and monitoring is handled and the analysis (seeing Table 1) of the blood sample that is untreated at one time.

Patient's blood does not have the B malignant change of cell, and expression has similar immunophenotype to change trend to B-CLL patient's blood, but change degree difference.Yet bone-marrow-derived lymphocyte and MHC II class positive cell compares very low with absolute number with the number in the B-CLL blood samples of patients relatively in these blood samples of patients.

After its blood was handled, the brother of two shortage B cells with the chain baby's Hypogammaglobulinemia of X was at CD3 ⁺The relative number aspect of cell shows different immunophenotypes and changes.Younger brother be 2 months big, not morbidity shows CD3 when handling his blood ⁺Cell relative number purpose slightly increases, and follows CD3 ^-CD19 ^-Cell relative number purpose reduces.On the other hand, the elder brother is 2 years old, and the state of an illness is very serious, and its number of expressing the antigenic activated T cells of DR is high relatively, and shows CD3 when handling his blood ⁺The minimizing of cell count.Do not adopt other mark to measure other immunophenotype that may occur and change, because from these two patients, obtain blood sample considerably less (table 2, ID 43/BD and 04/BD).

Patient 91 in the table 2 shows CD3 after processing blood ⁺Cell relative number purpose reduces, and it follows CD3 ^-CD19 ^-Cell relative number purpose increases.Yet, when analyzing other surface markers such as CD4 and CD8 (seeing Table 3), find to have in this patient's the blood CD4 of high relatively number ⁺CD8 ⁺Cell, this found before handling blood sample with the monoclonal antibody of DR antigen β chain, and these pairs positive cell significantly reduces after blood treatment.In addition, when analyzing other mark, find that the relative number of CD3+ cell increases (seeing Table 4).

When in equipment of the present invention, handling the bone-marrow-derived lymphocyte enrichment preparation that from the blood donors of health, obtains, show CD3+ cell relative number purpose and acutely reduce, and always follow CD19 with the monoclonal antibody of DR antigen β chain ⁺Cell relative number purpose reduces and CD19 ^-CD3 ^-Cell relative number purpose increases.These mark relative number purposes of further analysis revealed of carrying out with marks such as CD4 and CD8 reduce simultaneously.Yet, when handling the T lymphocyte enrichment preparation of same blood donors, do not show same change with same monoclonal antibody.

CD4 and CD8 group

T4 antigen is human immunodeficiency virus's a acceptor.The CD4 molecule combines at the B2 structural domain with MHC II class antigen, and this zone is similar to the antigenic CD8 binding site of I class.CD4 strengthens antigenic t cell responses with antigenic combination of II class, and CD8 also is like this with antigenic combination of I class.CD8 antigen is present on people's inhibition/cytotoxic T cell hypotype, and is present on the lymphocytic a kind of hypotype of NK cell (NK) and the most of normal thymus cell.CD4 and CD8 antigen coexpression are on thymocyte, and these cells lose one of mark when maturation is the T lymphocyte.

As can be seen, a kind of staining method is supported the reverse undifferentiated cell that is divided into of bone-marrow-derived lymphocyte, and is divided into the lymphocytic existence of T subsequently in most of blood samples that---seeing below---lists from table 2 when analyzing CD4 and CD8.

Two positive cell CD4 ⁺CD8 ⁺Cell always occurs after the monoclonal antibody processing blood with DR antigen β chain, and the cell of these types significantly increases in the treated blood sample of B-CLL patient, does not exist in untreated samples (see Table 3 and

chart

1,2,3 and 4).In identical sample, single positive cell such as CD8 have also been noticed ⁺And CD4 ⁺The relative number of cell increases simultaneously.In addition, at least under the situation of B-CLL corresponding to the CD4 of B cell ^-CD8 ^-Cell relative number purpose reduces in handling sample with in the untreated samples compares remarkable minimizing, when measuring in time, keeps par in the untreated samples.Yet, handle in the sample in time to CD8 ⁺CD4 ⁺The measurement that the relative number of cell carries out shows that the increase of single positive cell follows two positive cell relative number purposes to reduce.It is the feature (

patient number

2,3 and 4) of the thymus development of the progenitor cell of T lymphocyte series in the thymus gland that such immunophenotype changes.(CD4 on auxiliary/inducer T lymphocyte hypotype that T4 antigen is present in ⁺CD3 ⁺) and most of normal thymus cell on.Yet this antigen is at monocytic cell surface and monocyte and scavenger cell (CD3 ^-CD4 ⁺) in exist with low density.

Low CD4 ⁺The relative number of cell is being subjected to Different Effects after the device processes of the present invention in different blood samples.As if the relative number of the type cell unaffected in B-CLL patient's blood sample.This low-level CD4 expresses and is found in monocyte and very early stage thymocyte.

The HIV that handles ⁺25 patients show the increase largely of two positive cells of expressing CD4 and CD8 simultaneously.On the other hand, the patient 91 who treats shows the minimizing of this hypotype cell, and the observation of this phenomenon is a time-dependent manner.Find CD8 ⁺The relative number of cell increases in B-CLL patient's the blood sample that is untreated in time, finds CD4 simultaneously ⁺With low CD4 ⁺Cell reduces (table 3, the

patient

2,3 and 4).

DR and CD3 group

The DR mark is present in monocyte, dendritic cell, B cell and activated T lymphocyte.

The processing of carrying out with this group and the analytical table of untreated samples reveal to CD19 during with CD3 labeled analysis sample similar immunophenotype change (seeing Table 2), these aforementioned antigens are respectively full B cell and T cell marking.

In equipment of the present invention, as if influence DR with the monoclonal antibody processing blood ⁺The relative number of bone-marrow-derived lymphocyte, so DR ⁺Cell reduces.On the contrary, CD3 ⁺The relative number of (T cell) cell significantly increases (see Table 4 and chart).In addition, the relative number of activated T cells increases in most of treated B-CLL patient's blood samples, and the cell of these types is subjected to different influences in the treated sample of the patient with other situation.

In addition, exist with significant number in the relative number of the high positive cell of the DR 6 age in days babies' that the DR+CD34+ immature cell increases in B-CLL patient and blood the treated sample.Yet, should be noted that the immature cell that is present in this blood samples of patients is being negative for T cell and B cell marking before and after treatment, but be that the antigenic positive is stronger handling juvenile cell.In most of treated blood samples, CD3 ^-DR ^-The relative number of cell increases, and is directly proportional with the relative number increase of CD3+ cell (T cell), is reduced to inverse ratio with the relative number of DR+ cell (B cell).

CD56﹠amp; 16 and CD3 group

CD56﹠amp; The CD16 mark is present in one group of allos cell, a kind of large granular lymphocyte and lymphocytic lymphocyte subtype of NK cell (NK) of being called.The CD16 antigen presentation is in basic all tranquillization NK lymphocytes, and comes from some individual CD3 at some ⁺Weak expression on the T lymphocyte.Have been found that this antigen low scale on granulocyte reaches, and relevant with the lymphocyte that contains big azurophilic granule.CD16 antigen is IgG FC receptor II I.

The CD16 of various numbers ⁺Lymphocyte coexpression CD57 antigen or low density CD8 antigen or the two.In most of individualities, basic and other T lymphocyte antigen such as CD5, CD4, or CD3 antigen does not have overlapping.CD56 antigen is present in basic all tranquillization and activated CD16 ⁺NK lymphocyte, these cell subsets are carried out the cytotoxicity of non-main histocompatibility complex restriction.

The patient's of processing and untreated B-CLL and some other situations blood sample immunophenotype analytical table reveals expresses CD56﹠amp; The antigenic cell number of CD16 increases, and these cells have many particles, size medium (see Table 5 and

chart

1,2,3,4).These observationss are also followed cell and the coexpression CD56﹠amp that only expresses CD3 antigen (not expressing CD56 and CD16 mark); The relative number of the cell of CD16 and CD3 mark significantly increases.

In table 5, the

patient numbers

2,3, with the identical blood sample of 4 representatives, but are respectively to analyze (before and after treatment) at 2 hours, 6 hours and 24 hours.As if this sample shows that the monoclonal antibody processing blood with DR antigen β chain causes spontaneous generation CD56 ⁺And CD16 ⁺Cell, CD3 ⁺Cell and CD56 ⁺And CD16 ⁺CD3 ⁺Cell, these observationss are always followed B cell marking (CD19, DR, CD56, CD16 ^-CD3 ^-) disappearance.

Analyze expression, CD56 at the forward direction that before and after treatment this blood sample is carried out (onward) ⁺And CD16 ⁺Cell levels reduces in time, CD3 ⁺The level of cell increases in time.

Change with respect to observed immunophenotype in processing and the untreated samples, B-CLL patient 7 blood sample does not show expresses CD56, the antigenic cell number of CD16 and CD3 changes, and this is because the monoclonal anti scale of construction that adds is compared very low with the number of bone-marrow-derived lymphocyte.Yet the blood sample of handling the patient with an amount of monoclonal antibody under situation independently shows CD3 ⁺, CD56 ⁺﹠amp; CD16 ⁺And CD56 ⁺And CD16+CD3 ⁺The relative number purpose of cell significantly increases.

Blood sample with patient of other situation shows the various changes of these cell levelss, as if this depends on the number of handling bone-marrow-derived lymphocyte preceding, that exist when handling, and the clinical condition that may depend on this patient.

CD45 and CD14 group

CD45 antigen is present in all human leucocytes, comprises lymphocyte, monocyte, polymorphonuclear cell, eosinophil and basophilic cell in peripheral blood, thymus gland, spleen and the tonsilla, and the lymphocyte progenitor cell in the marrow.

CD14 is present in the normal peripheral blood mononuclear cells of 70-93%, the hydrothorax of 77-90% and ascites scavenger cell.This antigen weak expression on granulocyte, and be not present in lymphocyte, mitogen activated T cells, lymphocyte, red corpuscle or the thrombocyte that does not stimulate.

CD45 antigen is represented a kind of Protein-tyrosine-phosphatase, and this molecule and outside stimulus (antigen) interact, and influences the signal conduction by the Scr family member, causes the adjusting of cell growth and differentiation.

DR antigen β chain is at treated sample, and the participation in the sample that particularly obtains from B-CLL patient is pointed out, and this processing influences the antigenic level of CD45 on the bone-marrow-derived lymphocyte.As if the overall immune phenotypic alternation that occurs in the stimulation of DR antigen β chain produce dissimilar cells, they are based on the expression level of CD45 and CD14 and profile and assemble, profile determines that by direct scattering and lateral scattering (how much determining size and particle respectively) these results are shown in table 6 and chart (1,2,3,4).Also referring to Fig. 7, it has proved with CD45 after the CR3/43 antibody treatment ^-CD14 ^-The profile of cell.These cells are not hematopoietic cells.

With device processes of the present invention the time, the relative number (comparing with untreated samples) of low CD45 cell significantly increases, and the relative number of coexpression CD45 and the antigenic cell of CD14 also is like this.Such immunophenotype change takes place simultaneously with the relative reduced number (comparing with untreated samples) of high CD45 cell.Yet a kind of cell mass in back can further be distinguished according to the expression degree of profile and CD45.One type very big, compares the CD45 antigen with high level with other cell of representing in the chart (seeing

chart

1,2,3,4).Analyze this group (see Table 6, the

patient

2,3 and 4, and chart 1) after the processing in time, find that originally the relative number of CD45+ acutely reduces in time, to produce low CD45 cell.Yet 24 hours post analysis blood is found opposite situation.

It is opposite that sample 5 and 7 immunophenotype change the result who obtains with other sample that obtains from other B-CLL patient, and this is to be the more early stage analyses of carrying out of hatching with monoclonal antibody because of these samples.In fact, to handling the sequential analysis prompting of back blood sample, the immunophenotype change that bone-marrow-derived lymphocyte carries out is a time-dependent manner, because the stage of its representative growth, the immunophenotype of measuring at time point X changes different later on time point X (in case it is not just to induce to fix).Yet the change of these types must take place in stricter mode in health, otherwise immunological disease will take place.Never the treatment effect of the blood sample that obtains of other patient of B malignant change of cell is showing various changes aspect the immunophenotype of cell, and this is because bone-marrow-derived lymphocyte exists with low amount.Yet handling immunophenotype that the enriching section of the bone-marrow-derived lymphocyte that obtains from the healthy blood donor shows, to change the immunophenotype change that obtains with the B-CLL with the high counting of bone-marrow-derived lymphocyte similar.

CD8 and CD3 group

CD8 antigenic determinant and I class MHC interaction of molecules cause the adhesion between CD8+T lymphocyte and the target cell to increase.Such interaction has strengthened the lymphocytic activation of tranquillization.CD8 antigen and protein tyrosine kinase (p56ick) coupling, the CD8/p56ick mixture can work in the T lymphocyte activator subsequently.

In equipment of the present invention, handle the blood sample that obtains from B-CLL patient and cause CD3CD8 and CD3 (being likely CD4CD3) positive cell relative number purpose significantly to increase, clearly illustrate that therefore that the two positive cells that originally produce are being grown and be mature T lymphocyte with the monoclonal antibody of B chain.This is one can directly measure and use CD8 with CD19 and DR ^-CD3 ^-The process that antigen indirect is measured.Assess as if consistent with the process that is equal to thymic cell development (table 7, the

patient

2,3 and 4, and chart 1) with the series in time that the treated blood sample of same patient is carried out.

The relative number of CD8+ cell increases in processing and untreated samples in time, but increases to higher degree in untreated samples.On the other hand, CD8 ⁺CD3 ⁺The relative number of cell reduces in untreated samples in time.Yet, when measuring in time, handle CD3 in the blood sample ⁺The relative number of cell increases, and the cell height of these types is corresponding to CD4 ⁺CD3 ⁺Single positive cell, i.e. a kind of thymocyte of mature form.In addition, because these samples have also carried out the immunophenotype analysis with other group (mentioning) in table 3,4,5 and 6, overall change shows that strongly the B cell participates in T lymphocyte ancestors and offspring's generation.

Blood sample (2,3 and No. 4, table 1,2,3,4,5,6,7) the five equilibrium thing that B-CLL patient is obtained does not process, or handles with the non-coupling form of the monoclonal antibody of PE link coupled DR antigen β chain homologous region and same monoclonal antibody.Relatively the PE link coupled is handled and is clearly show, the relative number of CD3 positive cell and other mark of correlation such as CD4 does not change, and has observed conspicuous level when same blood sample is handled with the non-coupling form of antibody.Yet, find that when measuring in time CD45 positive cell number purpose increases, these cell surfaces do not have DR antigen presentation (seeing Table 8).Carry out in untreated samples, having noticed when immunophenotype is analyzed similar result's (table 6) when in time.And the relative number of expressing the cell of low CD45 reduces in time, and (when measuring in time) also noticed this phenomenon (seeing chart 1A) in same patient's untreated samples.

The FAC that comes from the cell of people's buffy coat sample analyzes

Handle the buffy coat sample with the CR3/43 monoclonal antibody and cause the occurrence rate of CD34 (stem cell labeling) higher in equipment of the present invention, the occurrence rate of CD19 (bone-marrow-derived lymphocyte mark) is lower---see Table 21.

Colony forming assay is found in untreated samples, most colonies are red corpuscle types, and the change of colony type is more obvious in handling sample, and main colony type is the pluripotent cell colony, goes back granulocyte/scavenger cell and megalokaryocyte and CFU-E.

These results prove that treated cell more can produce each species specific clone along other lymph hematopoiesis approach differentiation.

C. relatively the T lymphocyte is generated and have the not effect of homospecific other monoclonal antibody

CD19 and CD3 group

In equipment of the present invention, handle blood sample and reduced CD3 with the monoclonal antibody of DR antigen α chain homologous region and MHC I class antigen homologous region ⁺The number of cell has increased CD19 ⁺The number of cell.Handle blood sample with the monoclonal antibody of DR antigen β chain homologous region and reduced CD19 ⁺The number of cell has increased CD3 ⁺The number of cell.Handle the same effect (table 14, B-CLL patient 5/6, handles back 2 hours) of having found with a kind of monoclonal antibody in back and endoxan.

The CD19 that in same sample, carries out ⁺And CD3 ⁺The forward direction analysis revealed of cell, CD3 ⁺The relative number of cell only further increases (table 14, B-CLL patient 5/6, handles back 24 hours) in the blood of handling with the monoclonal antibody of DR antigen β chain homologous region.Yet, the forward direction analysis (table 14, B-CLL patient is after 5/6,24 hour) that adds the blood sample that the monoclonal antibody of DR antigen β chain homologous region handles with endoxan show with similarity condition under under 2 hours incubation time observed CD19 ⁺And CD3 ⁺The relative number of cell is opposite.

Generally speaking, compare, handle the relative number that blood sample has increased CD19+ cell (full B mark) with the monoclonal antibody of DR antigen α chain homologous region or the monoclonal antibody of MHC I class antigen α chain homologous region with untreated samples.CD19 in the blood sample of handling with the monoclonal antibody of DR antigen α chain or the antigenic monoclonal antibody of I class ^-CD3 ^-The relative number of cell reduces (see Table 14 and

chart

2,3,4) slightly.The blood sample of handling patient 09 with the antigenic monoclonal antibody of I class has increased the relative number of CD3+ cell, has reduced CD19 slightly ⁺And CD19 ^-CD3 ^-The relative number of cell.Yet, handle the immunophenotype that the bone-marrow-derived lymphocyte enrichment preparation that obtains from the blood donors of health shows with the monoclonal antibody of DR antigen α chain or β chain and change similar with the immunophenotype change that obtains from B-CLL patient.

Monoclonal antibody with DR antigen β chain is handled HIV ⁺Increased CD3 with IgA defective patient ⁺The relative number of cell has reduced CD19 ⁺The relative number of cell.Yet, handle same blood sample with the monoclonal antibody of I class antigen homologous region and do not produce identical effect.Monoclonal antibody with DR antigen β chain, I class antigen and T4 antigen is handled the blood sample that lacks patient (34/BD and 04/BD) acquisition from the B cell, has shown various immunophenotypes and has changed.

CD4 and CD8 group

Also the blood sample that carried out analyzing with CD19 and CD3 group (table 19) is carried out immunophenotype branch (table 15) with CD4 and CD8 group.As if this result of two groups consistent.Monoclonal antibody or same monoclonal antibody with DR antigen β chain homologous region add the blood sample 2 hours (table 15, patient 5/6 and 10,

chart

2,3 and 4) that endoxan is hatched B-CLL patient, have increased CD8 ⁺And CD4 ⁺The relative number of the cell of two kinds of marks of cell and coexpression.On the other hand, handle same sample with the monoclonal antibody of DR antigen α chain homologous region or I class antigen α chain homologous region and do not produce same effect.

Relatively add endoxan and hatch the immunophenotype trend that obtains after 2 hours and 24 hours with the monoclonal antibody of DR antigen β chain, find that CD4 and CD8 positive cell relative number purpose change (table 15 on the contrary, B-CLL patient 5/6,2 hours and 24 hours), this change and the unanimity as a result that is obtained with CD19 and the same blood sample of CD3 group analysis (table 14, the patient is identical).Back one is found to show that follow-up differentiation is a reversible, because undifferentiated cell can be divided into T lymphocyte or bone-marrow-derived lymphocyte.

DR and CD3 group

The immunophenotype that obtains with DR and CD3 (table 6) group changes the result that confirmed to be obtained by CD19 and CD3 group and CD4 and CD8 group (table 14 and 15 and

chart

2,3 and 4), this result is monoclonal antibody or the antigenic monoclonal antibody of I class with DR antigen α or β chain homologous region, or the monoclonal antibody of DR antigen β chain homologous region adds endoxan and handles that 2 hours analyze behind the same blood sample.

Find that from this result the monoclonal antibody of DR antigen β chain homologous region can cause from DR very much ⁺Cell produces the CD3 positive cell.

In addition, the processing of uniting participation (incubation time of prolongation) that for example relates to DR antigen α chain or DR antigen β chain and endoxan has promoted CD19 ⁺Cell or DR ⁺Cell relative number purpose increases.

CD56﹠amp; 16 and CD3 group

Handle blood sample with the monoclonal antibody of DR antigen β chain homologous region in equipment of the present invention, especially handling the B-CLL patient with the high counting of bone-marrow-derived lymphocyte has increased CD56﹠amp; The relative number of 16 positive cells.

In these patients, after handling blood sample, also increased CD3 with the monoclonal antibody of β chain ⁺And CD56 ⁺And CD16 ⁺CD3 ⁺The relative number of cell, observed result when having confirmed in early stage same the processing with the same blood sample of CD3 and CD19 and DR and CD3 group analysis.

CD45 and CD14 group

Blood sample after adding endoxan or the antigenic monoclonal antibody of I class and handle with the monoclonal antibody of the monoclonal antibody of DR antigen β or α chain or β chain in equipment of the present invention is also organized with CD45 and CD14 and has been carried out analyzing (table 18).Qualification to low CD45, high CD45 and medium CD45 is authoritative.Add the low CD45 of endoxan processing blood sample 5/6 (at 2 hours) generation with the monoclonal antibody of DR antigen β chain or with this monoclonal antibody ⁺Cell also increases medium CD45 ⁺The relative number of cell.Yet preceding a kind of processing has increased high CD45 ⁺The relative number of cell, a kind of processing in back has reduced medium CD45 ⁺The relative number of cell, these change time-dependent manner seemingly.

The patient's 5/6 and 10 (B-CLL) who handles with the antigenic monoclonal antibody of I class blood sample shows medium CD45 ⁺Cell relative number purpose reduces, blood sample 09 and HIV after same the processing ⁺In compare with untreated samples and also to have noticed the similar viewing result.Compare with untreated samples or with the monoclonal antibody processing of DR antigen β chain, the blood sample of handling HIV+ and IgA/D patient with the antigenic monoclonal antibody of I class has increased low CD45 ⁺The relative number of cell.Yet these patients' blood sample shows medium CD45 after handling with the monoclonal antibody of DR antigen β chain homologous region ⁺Cell relative number purpose reduces.After the antigenic monoclonal antibody of I class is handled, the medium CD45 in IgA/D patient's blood sample ⁺Cell increases.In the blood sample of handling with the monoclonal antibody of the antigenic DR antigen of MHC II class β chain homologous region, noticed very large, particle is very many, express the antigenic cell of high-level CD45 (seeing

chart

1,2,3,4).

CD8 and CD28 group

CD28 antigen is present in peripheral blood T (CD3+) lymphocyte of about 60-80%, 50% CD8 ⁺T lymphocyte and 5% prematurity CD3 ^-Thymocyte.In the thymus gland ripening process, the CD28 antigen presentation is from most of CD4 ⁺CD8 ⁺Low density on the prematurity thymocyte increases to nearly all ripe CD3 ⁺, CD4 ⁺Or CD8 ⁺More high-density on the thymocyte.The expression of CD28 is also with CD8 ⁺Lymphocyte is divided into two functional group.The specific cytotoxicity of CD8+CD28+ cell mediated allogenic antigen, this is the main histocompatibility complex of I class (MHC) restriction.The inhibition of cell proliferation is by CD8 ⁺CD28 ^-The hypotype mediation.CD28 antigen is a kind of cell adhesion molecule, as the antigenic part of B7/BB-1 that is present on the activated bone-marrow-derived lymphocyte.

Monoclonal antibody treatments B-CLL patient (table 19, patient 5/6 and 8) with DR antigen β chain homologous region in equipment of the present invention has increased CD8 ⁺, CD28 ⁺And CD8 ⁺CD28 ⁺The relative number of cell, the processing of other type does not then have.

CD34 and CD2 group

CD34 antigen is present in all the hematopoiesis colony forming cells in jejune hemopoietic forebody cell and the marrow, and (CFU-GM is BFU-E) with multipotency ancestors (CFU-GEMM, CFU-Mix and CFU-blast) to comprise monoenergetic.CD34 also expresses on the stroma cell precursor.In normal bone, terminal deoxyribotide transferase (TdT) ⁺B and T lymphocyte precursor are CD34 ⁺, CD34 antigen is present in expresses CD33 antigen but shortage CD14 and the antigenic early stage medullary cell of CD15, and expresses CD71 antigen and the antigenic early stage red corpuscle of faint expression CD45.CD34 antigen also is present in the human thymocyte of capillary endothelial cell and about 1%.Normal circumference blood lymphocyte, monocyte, granulocyte and thrombocyte are not expressed CD34 antigen.The CD34 antigen density is the highest on the hemopoietic progenitor cell in early days, reduces when cell maturation.On the hematopoietic cell of differentiation fully, there is not this antigen.

Unshaped CD34 ⁺Progenitor cell is CD38 ^-, DR ^-And lack the clone specific antigens, and as CD71, CD33, CD10, and CD5, and CD34 ⁺Cell is the clone typing, expresses CD38 antigen with high-density.

Most of CD34 ⁺Cell is expressed CD45RO or CD45RA antigen mutually.About 60% acute B Lymphocytic leukemia and acute myelocytic leukemia are expressed CD34 antigen.This antigen is not expressed on chronic lymphocytic leukemia (B or T clone) or lymphoma.CD2 antigen is present in a kind of hypotype of T lymphocyte and NK cell lymphocyte (NK).

The results are shown in

chart

2,3 and 4.

Analyze the blood sample (table 20, patient 5/6,2 hour) with the monoclonal antibody treatments B-CLL patient of DR antigen β or α chain, find that CD34+ relative number purpose significantly increases, after preceding a kind of antibody treatment, the relative number of CD34+CD2+ cell significantly increases.Owing to identical blood sample has been carried out the immunophenotype analysis, observed CD34 here with above-mentioned each group (seeing Table 14-19) ⁺And CD34 ⁺CD2 ⁺As if cell relative number purpose increase and CD4 ⁺CD8 ⁺, CD8 ⁺CD3 ⁺And CD4 ⁺CD3 ⁺Single positive (SP) cell relative number purpose increases consistent.In addition, as if these discoveries that relate to DR antigen β chain specially support this process to cause the T lymphocyte to generate by the bone-marrow-derived lymphocyte degeneration indirectly.

When analyzing same processing, as if the CD34+ cell reduces to certain level after 24 hours, thereby cause T lymphocyte relative number purpose further to increase.Originally this reverse atomization causes the T lymphocyte to generate, and this process can be inverted, and causes bone-marrow-derived lymphocyte to generate.Before a kind of phenomenon be that to add endoxan 2 hours in the monoclonal antibody with DR antigen β chain viewed, then a process is (chart 2) noticed 24 hour incubation period when same sample being carried out same processing.

The blood sample of handling HIV+ patient with the monoclonal antibody of DR antigen β chain has significantly increased the relative number of CD34+ and CD2+CD34+ cell, and the identical blood sample of handling with the monoclonal antibody of the monoclonal antibody of the HLA-DR antigen β chain that adds simultaneously and same antigenic α chain also is like this.Yet handling this blood sample with the monoclonal antibody of HLA-DR antigen α chain does not influence CD34 ⁺The level of cell.With the monoclonal antibody of the monoclonal antibody of HLA-DR antigen β chain and same antigenic α chain with add two kinds of monoclonal antibodies simultaneously and handle the blood sample that obtains from one 6 age in days baby (BB/ST table 20), produces following immunophenotype change.

When blood sample is untreated in analysis, CD34 ⁺And DR ⁺The relative number of cell significantly increases, when handling with the monoclonal antibody of β chain, and CD34 ⁺The relative number of cell further increases, but handles or this reduced number during with the monoclonal antibody processing of the α chain of this molecule that adds together and β chain with the monoclonal antibody of HLA-DR antigen α chain.Yet a kind of processing in back has increased CD34 ⁺CD2 ⁺The relative number of cell produces opposite result when independent monoclonal antibody with HLA-DR antigen β chain is handled.As 24 hours same patients of post analysis during branch thing such as treated or untreated blood, above-mentioned all handle and all make CD34 ⁺Relative reduced number, kept higher level when removing the monoclonal antibody processing with HLA-DR antigen β chain.A kind of processing in back continued to reduce CD34 after 24 hours ⁺CD2 ⁺The relative number of cell.

These results show, HLA-DR antigen has promoted from B-CLL patient's CD2+CD34+ pond by relating to of β chain or more sophisticated cell type such as bone-marrow-derived lymphocyte produces more CD34 ⁺Cell, these results show that such processing has promoted reverse differentiation.Yet, the immunophenotype analysis of blood sample prompting after 24 hours, as if these cell types all be present in another kind of clone, and as if cell is present in or it is finalized the design in myelocytic series in this case, and this is using CD7 and CD13﹠amp; Be observed in the analysis of the 33 treated blood samples that carry out.

When the bone-marrow-derived lymphocyte of monoclonal antibody treatments B-CLL that uses MHC II class antigen β chain homologous region and healthy individual enrichment (using the CD19 pearl) part, profile has changed its immunophenotypic characteristics.These have followed the change of bone-marrow-derived lymphocyte profile.Observing the bone-marrow-derived lymphocyte that forms colony in the blood smear that is untreated on slide is replaced by the cell of granulocyte, monocyte, a large amount of original forms and erythroblast.On the processing or the blood smear that is untreated, do not observe mitotic division feature or significant necrocytosis.

The result of table 20 has also further proved an important discovery, and promptly the method according to this invention can prepare undifferentiated cell by making the reverse differentiation of more sophisticated undifferentiated cell.

D. microphotograph

Except above-mentioned antigen check, also use microscope from visually abideing by method of the present invention.Can programme to equipment of the present invention, it is changed from motion tracking by trace tool.

In this, Fig. 8 is the microphotograph of differentiation B cell before method of the present invention.Fig. 9 be the method according to this invention by the reverse microphotograph that is differentiated to form undifferentiated cell of B cell, wherein this reagent is the monoclonal antibody of HLA-DR antigen β chain homologous region.Undifferentiated cell is the cell mass that secretly dyes.Figure 10 is the microphotograph of same undifferentiated cell, but magnification is lower.

Therefore Fig. 8-10 is from visually having proved by method of the present invention the reverse undifferentiated stem cell that is divided into of B cell.

Figure 11 is the microphotograph of the differentiation B cell before the inventive method.Figure 12 be the method according to this invention by the reverse microphotograph that is differentiated to form undifferentiated cell of B cell, use therein reagent is the monoclonal antibody of HLA-DR antigen β chain homologous region.Equally, undifferentiated cell is the cell mass that secretly dyes.Figure 13 is the granulocytic microphotograph of identical undifferentiated cell formation differentiation from Figure 12.

Therefore Figure 11-13 is from visually proving by method of the present invention the reverse undifferentiated stem cell that is divided into of B cell, then undifferentiated cell is fixed to new and the not homologous noble cells of original noble cells.

Also the blood sample to the B-CLL patient that handles with the CR3/43 monoclonal antibody has carried out the microscope experiment, and as previously discussed, the blood of BCLL can be used for helping the reverse atomization of research, because the bone-marrow-derived lymphocyte that this blood contains is than normal number height.This result is expression in detail in Figure 14-17.

Figure 14 is illustrated under two kinds of different magnifications, the blood sample that is untreated that obtains from BCLL patient.The bone-marrow-derived lymphocyte (blue cell) that is untreated shows representative configuration, promptly spissated chromatin Structure and rare tenuigenin.Remaining cell is a red corpuscle.

Handling blood sample with antibody CR3/43 causes the bone-marrow-derived lymphocyte of cluster to assemble (Figure 15) at the beginning.

The B cell of cluster loses their representative configuration gradually, it is characterized in that forming pebbles like cell zone, and the going of chromatin Structure concentrates the appearance of typical kernel, the typical tenuigenin basophilia of the expansion of cell volume and undifferentiated cell (Figure 16).(the going to concentrate) chromatin Structure of loosening is a kind of key character that undifferentiated cell is compared with noble cells.This may be since need more extensive near transcription unit, to determine along the finalize the design change of needed genetic expression of given clone.On the contrary, known more noble cells has more spissated chromatin Structure, because only need a small amount of chromatin to have transcriptional activity.

The appearance (17A-17J) of the cell with differentiation profile is always followed in the appearance of undifferentiated cell.Importantly, these cells can not be accelerated by propagation, this is because (i) incubation time is too short for the complete cell fission of one or many takes place, and does not (ii) see the mitotic division feature, and (iii) identical with leukocytic absolute number maintenance afterwards before handling.In addition, find that the ancestors that more do not break up are relevant with their more differentiation offspring (seeing the bone marrow precursors of Figure 17 J), this shows that these special cells produce by differentiation.

Displaing micro photo figure 17A-17J represents the type with the noble cells of being seen behind CR3/43 monoclonal antibody treatments B-CLL lymphocyte: thrombocyte (PI)-Figure 17 A, neutrophil leucocyte (Ne)-Figure 17 B, eosinophil (Eso)-Figure 17 C, megalokaryocyte (Meg)-Figure 17 D, basophilic cell (Ba)-Figure 17 G, lymphocyte (Ly)-Figure 17 H, monocyte (Mo)-Figure 17 I and myeloid progenitor (Mp)-Figure 17 J.Red corpuscle ancestors and scavenger cell (not providing data) have also been seen.

So in a word, these profiles result represents the change of B cell appearance in BCLL patient's sample, has high-caliber ripe bone-marrow-derived lymphocyte among these patients.The profile that microphotograph has showed bone-marrow-derived lymphocyte changes, they at first assemble cluster, occur various cells (neutrophil leucocyte, basophilic cell, eosinophil, megalokaryocyte, thrombocyte, lymphocyte, scavenger cell, granulocyte, thorn granulocyte and matrix like cell) with the fractionated profile scope from progenitor cell to noble cells then.

In addition, and it is highly important that, seen red corpuscle and myelocyte ancestors (Figure 17 J-data not shown goes out).This myelocyte ancestors can be by profile clearly with other cell differentiation, and they are bigger, have unique nuclear morphology, and contain the kytoplasm particle.

Therefore, the microscope data are supported the flow cytometry data represented with cell surface marker from profile.These data allow to reach a conclusion, and promptly causing other hematopoiesis such as the minimizing of bone-marrow-derived lymphocyte number and prematurity precursor cell with the antibody treatment bone-marrow-derived lymphocyte of MHC HLA-DR β chain is the increase of cell number.

Also with microscopic examination the reverse undifferentiated stem cell that is divided into of T cell that the antibody (monoclonal antibody TAL.1B5) that adopts MHC II class α chain is handled by method of the present invention, then undifferentiated cell is fixed to new noble cells, this cell and primary noble cells be homology (data not shown goes out) not.

E. analyzing the VDJ reorganization in reverse differentiated lymphoid resets

By background, the noble cells (bone-marrow-derived lymphocyte or the cell with some T lymphocyte feature) that is used for these experiments has to be reset and the gene of difference encoding mature Ig or TCR.In rearrangement process, not as the DNA middle portion of a final part, the TCR that is expressed or Ig gene, promptly the original DNA between the district's encode fragment of variable (V) between these acceptors and constant (C) district encode fragment is excised from genome.These cut fragments exist with the form of DAN outside the karyomit(e) in cell.For the cell of real reverse differentiation, cut DNA will insert genome again, make cell be in to their original differentiation before similar state.Because this point, compare with rearrangement DNA as the feature of differentiation state, do not reset or during the reproductive tract state when DNA turns back to it, be complementary to reset the sequence in the gene probe will with bigger DNA restricted fragment hybridization.

1.Daudi the rearrangement of tcr gene in the cell

In the experiment that produces southern blotting technique shown in Figure 180, adopted known clone Daudi, promptly a kind of B cell lymphoma with rearrangement tcr gene (another is removed).From Daudi cell preparation genomic dna, and, carry out gel electrophoresis, survey with the TCR β ssdna probe of mark then with EcoRI digestion.Adopt the Daudi cell, rather than from patient the bone-marrow-derived lymphocyte of purifying, it is relevant that this is because of these cells that the clone goes up, and come from and have the isogenous group that homologous genes is reset, and this can clearly observe by the southern blotting technique of the genomic dna that digested.In normal blood sample, different cells has different rearrangements, so southern blotting technique will occur as smear.

The functional gene of coding TCR β chain is assembled in lymphocyte, and this is to reset by a series of somatic chromosomes in the lymphocyte ripening process to form, and this rearrangement flocks together V fragment, D fragment and J fragment.The university textbook of a standard is seen in the explanation that is perfectly clear of these rearrangement process: Genes VI, Lewin, Oxford Universiy Press.1997 (P 1994-1023).Its some specific page have been quoted below.

At first, the D fragment is connected with some J fragments in the D-J ligation by regrouping process.Then, one of many possible V fragments (＜60) are connected in the DJ fragment (V-D connection) that obtains, so that form a complete TCR β chain gene.The constant region gene is in the downstream of resetting VDJ fragment next-door neighbour, but has the J of interleaving fragment, and they are cut in the RNA course of processing, make the constant region gene extron be adjacent to rearrangement the VDJ gene fragment (Lewin, p998).

In the human cell, two kinds of different TCR β chain constant region gene fragments are arranged, be called C β 1 and C β 2, they are present in two different locus, each front all has cluster 6 or 7 joining regions (J β) gene fragments (J β 1 and J β 2) and a D fragment (D β 1 and D β 2) (to see Fig. 1, Toyonaga et al., 1985, Proc.Natl.Acad.Sci.USA82:8624-8628 and Lewin, p1017).

Cause V, the contiguous recombination event of D and J-C fragment is catalytic by a plurality of albumen, comprises RAG-1 and RAG-2, and their identification is present in V, nine aggressiveness and the heptamer sequence of D and J-C gene order reorganization end.According to the direction of these nine aggressiveness/heptamer sequence, reorganization causes oppositely or removes.Two kinds of incidents all cause the change of genomic dna restriction enzyme fragment schema.In addition, removal incident might not cause cut segmentally lose fully.Cut segmental end can be reconnected, and produces to be retained in DAN ring (Okazaki et al., 1987, Cell49:477-85 in the cell; Davis et al., 1991, J.Exp.Med.173:743-6; Livak and Schatz, 1996, Mol.Cell Biol.16:609-18; Harrimanet al.1993, AnnuRev Immunol.11:361-84).Certainly, every kind of gene fragment all has two allelotrope, because cell has the diploid chromosome complementation.

Normal the kind is under the state, and C β 1 and C β 2 genes are according to Toyonaga et al., the arrangement shown in Figure 1 in 1985.With EcoRI genomic dna is carried out restrictive diges-tion and will produce two relevant bands, they can be by the probe in detecting that adopts in the experiment to (this probe is the dna fragmentation that derives from C β 1 of mark, because height sequence homology, they are also hybridized with C β 2), these two bands are: the 12kb band that (i) contains C β 1 sequence; The 4kb band that (ii) contains C β 2 sequences.This kind is that configuration can be seen (Figure 18 A) in undifferentiated immature cell.This kind is that configuration has also carried out complete explanation by the 3rd road (using CR3/43 antibody, 2 hours) of Figure 18, produces the pattern identical with the A road.

Under differentiation state, two allelotrope of C β 1 and C β 2 are rearranged, and are not 12kb fragments just at C β 1 locus like this, are not 4kb fragments just at C β 1 locus perhaps.In fact, on gel, there is not the hybridized fragment (this is because removed hybridization sequences as the result who recombinates from two C β 1 allelotrope) that derives from C β 1 locus.For C β 2 locus, in fact two main bands are arranged now corresponding to resetting the difference " allelotrope " that obtains on two karyomit(e)s.Maximum band is less than 4kb, corresponding to two fragments of resetting one of allelotrope.Nethermost band is that another resets allelic fragment.The little band of intermediary may come from have the different Daudi cells of resetting subclone---so it exists with allelic any one inferior molar weight.Yet the rearrangement state most clearly is shown in the 1st road, and wherein two main bands all are perfectly clear.

Handle with the negative control antibody that is incorporated into MHC-DR α chain (TAL.1B5) and in fact to cause top band to disappear in 2 hours, and nethermost band has identical intensity (seeing the 2nd road) with untreated cell in the 1st road.To this possible explanation be cell in differentiation, further cause another recombination event at allelic C β 2 locus, this causes the forfeiture of C β 2 sequences.This and known phenomena in full accord.

Handle 3 bands (seeing the 4th road) that as if recovered in untreated cell, to see in 24 hours with negative control antibody.Yet these bands are in fact than the lower position of being seen in the 2nd road of having moved to.Why not clear this is.Possible explanation is reintegrating of the sequence that taken place to have removed, with decyclization-excise-reintegrate model (Malissen et al., 1986, Nature 319:28-32) unanimity.Yet, do not observe this result with TAL.1B5 antibody at 2 hours or 24 hours, illustrate to kind to be that pattern is reset.In fact on behalf of the antibody of negative control-α chain, the 2nd and 4 roads do not cause planting is the recovery of sequence.

On the contrary, handling with the monoclonal antibody (CR3/43) of MHC-DR β chain that the result who obtained in back 2 hours shows corresponding to kind is the pattern of configuration, i.e. 12kb band and 4kb band (relatively the 3rd road and A road).In other words, these results represent that the kind of allelic C β 1 and C β 2 locus is that unrestricted model has obtained recovery.

We reach a conclusion as a result from these, and the band pattern of seeing in the 3rd road is represented the rearrangement of noble cells genomic dna, are configuration to produce kind again.

The importance of this discovery should be not out in the cold.Can reverse comprising the removal genome rearrangement, make genome return to atomization state before takes place.Most probable explanation is to reset oppositely being reversed of causing by C β 2 allelotrope in the differentiation, and the removal that causes losing C β 1 sequence of 12kb band also is reversed.The interior additional shape cyclic DNA that exists of removal incident center that the source that C β 1 sequence disappears is seemingly initial.The existing in of this cyclic DNA (seeing the reference of quoting) on the books in the prior art.Yet this taking place all is that genome recovers that cutter system is unimportant really, and importantly it has taken place really.

Monoclonal antibody (CR3/43) with MHC-DR β chain in equipment of the present invention continues to hatch, and has produced more complicated band pattern (the 5th road).Yet these bands are not represented the same strap in the untreated control.Specifically, still there is fragment (" C β 2 allelotrope ") with the about 12kb of probe hybridization.In addition, importantly understanding indicates the band of " C β 2 allelotrope " and does not correspond to observed band less than 4kb in the untreated control (the 1st road).Observed result's most probable explanation is that rearrangement process has for the second time taken place in the 5th road, because crossing pattern is similar to the tcr gene (cell that this explanation and flow cytometry data consistent, this data sheet reveal the characteristic cell marking with T cell increases) that wherein T cell is characterised in that rearrangement.Yet,, support to expose in 2 hours the result in the 3rd road, back being exposed to result that CR3/43 antibody sees in the 5th road after 24 hours no matter how definite molecule explain.

2.B-CLL the rearrangement of Ig gene in the cell

Obtained the southern blotting technique shown in Figure 19 B with chronic lymphocytic leukemia (B-CLL) patient peripheral blood cells.From these a large amount of mono-clonal B cell preparation genomic dnas,, carry out gel electrophoresis, and survey with the TCR dna probe of mark with BamHI and HindIII digestion.Handle these B-CLL cells with CR3/43 as described herein (the II class MHC chain of anti-HLA-DR, DP and DQ).Survey this trace with radio-labeling Ig J region probe.Two bands that obtain from untreated cell in the A road are represented the Ig allelotrope (father source and source of parents) of two rearrangements.These bands do not occur in the B road, and B represents in the road with the pattern of antibody treatment cell after 24 hours.Having occurred planting in their position is the characteristic 5.4kb band of Ig gene.

In another experiment shown in Figure 19 A, pair cell is not handled, or handles with the anti-II class MHC β indicated time of chain antibody.(left-half of gel) is by pcr amplification Ig VDJ district in the B-CLL cell of differentiation (contrast) and antibody treatment.This is from having produced the VDJ amplified production for handling cell.Yet, in the cell of antibody treatment, do not observe such band, because as the genomic dna that inserts through excision, " planting system " DNA configuration is difficult for the pcr amplification that the specific primer through benefiting from VDJ carries out.Similar experiment (gel right side) allows me to observe contrast, the behavior of the house-keeping gene of the β Actin muscle of promptly encoding.No matter how handle, β Actin muscle pcr amplification product does not have difference.Therefore as if, be somebody's turn to do the influence that " contrast " gene is not subjected to reverse atomization, this process makes the Ig gene with like cell produce very big change under similarity condition.

Result above-mentioned represents can induce with the agent treated cell that relates to suitable cell surface receptor the reverse differentiation of these cells, and this confirms (and monitoring) by the driving in the wrong direction of characteristic karyomit(e) DAN rearrangement of observing differentiation state on molecular level.Therefore, on the basis of molecular genetics and profile evidence, can reach a conclusion, use the cell of reagent (mAb) the treatments B lymphocyte series that relates to II class MHC β chain can make its reverse differentiation.On the contrary, induce reverse differentiation with the same cell of agent treated that relates to II class MHC α chain dissimilarly.As if they along B cellular pathways differentiation (forward).

F. to the further research of the reverse differentiation of bone-marrow-derived lymphocyte

As stated above in equipment of the present invention with the BCLL cell of CR3/43 antibody treatment BCLL patient's FACsVantage purifying (95% pure B cell), handle cell with flow cytometry.Below Table A in the result that represents further confirmed the result that obtains previously.The number of cell with characteristic cell surface marker (CD19, CD20 and CD22) of bone-marrow-derived lymphocyte system significantly reduces, and has occurred CD34 simultaneously ⁺The remarkable increase of cell number.The more important that should be noted that from Table A is, is that CD34 cell number negative and the clone feminine gender increases simultaneously.These undifferentiated cell indefinite forms are hematopoiesis system, in differentiation prior to CD34 ⁺Stem cell.In addition, by the light microscopy sample, find that many adherent cell types have the resemblance of non-hematopoietic cell.

Table A

Mark	0 hour	2 hours	24 hours
Mark	0 hour	2 hours	24 hours	CD20	73	67	16
CD14	0	3	23	CD20	73	67	16
CD14	0	3	23	CD34	0	1	23
CD7	0	2	0	CD34	0	1	23
CD7	0	2	0	CD16	8	3	2
CD19	95	71	1	CD16	8	3	2
CD19	95	71	1	CD22	5	3	2
CD33	0	0	0	CD22	5	3	2
CD33	0	0	0	CD3	0	0	0

Also proved to lose the CD19 mark, followed the CD34 cell surface marker on same cell, to occur, and with the Laser Scanning Confocal Microscope instant recording on video-tape.Add CR3/43 monoclonal antibody bone-marrow-derived lymphocyte before and dye green with the monoclonal antibody of FITC link coupled CD19.After adding the CR3/43 monoclonal antibody, cell has lost their green fluorescence, begin to dye redness with the monoclonal antibody of PE/Cy5 (or quantum is red) link coupled CD34, rather than green (seeing Figure 23, two still images that its expression obtains from the time lag video recording).This result proves that clearly in the reverse differentiation of B cell, clone specific marker such as CD19 disappear, and stem cell labeling such as CD34 express again.

G. induce reverse other reagent that is divided into hemopoietic stem cell of bone-marrow-derived lymphocyte

Originally in fact research identified three kinds of reagent---granulocyte/monocyte G CFS (GM-CSF), erythropoietin and mAb CR3/43.In equipment of the present invention,, with above-mentioned CR3/43 and the described method of TAL.1B5 are handled normal bone-marrow-derived lymphocyte preparation enrichment, purifying, and press the sample that foregoing was handled with the flow cytometry inspection with one of these three kinds of reagent.Compare with negative control, all three kinds of samples of handling with GM-CSF, erythropoietin or mAbCR3/43 all show and the consistent change of reverse differentiation.Specifically, all three kinds of reagent have all increased CD34 in the cell mass ⁺The relative number (seeing Figure 20) of cell.Yet, observed maximum effect with CR3/43, therefore, the more detailed research of selecting this reagent to be used for mentioning here.

H. the characteristic of the hemopoietic stem cell that produces by reverse atomization

Colony forming assay

In order to confirm to have the characteristic of not breaking up hematopoietic cell, the blood sample of handling with the antibody (CR3/43-sees above) of II class MHC β chain is carried out colony forming assay by observed CD34+ cell of flow cytometry and the undifferentiated cell by microscopic examination---be the standard method that is used to assess the primitive hematopoietic cell ability as known in the art.This colony forming assay can be carried out in equipment of the present invention automatically, as the part of follow-up mechanism.

The external colony of hemopoietic stem cell is measured and is allowed the former progenitor cell that has propagation, differentiation and grow for phenotype and sophisticated myelocyte of function and/or erythrocytic ability is carried out quantitatively.For example, in the presence of somatomedin, when stem cell is planted/be fixed in the soft gel matrix, can carry out external colony growth (propagation) and differentiation.

Figure 21 is that the colony of the stem cell of method generation of the present invention is measured, and adopts anti-phase bright-field microscope.In this measures, handle the B cell that obtains from the buffy coat of healthy blood donor with CR3/43mab, measure by material and the described colony that carries out of method part then.

(a)-(e) explanation of Figure 21:

A) to show red corpuscle, medullary cell and mixing (being made up of ripe medullary cell and red corpuscle) colony in the observed culture dish of 3 times of amplifications of bright-field microscope, they observe with the naked eye easily.Every kind of colony is produced by single hemopoietic stem cell by propagation and differentiation subsequently.

B) MIX-CFC, this colony result from single pluripotential hemopoietic stem cell (can produce the stem cell of medullary cell and erythron cell).

C) M-CFC, this colony is made up of scavenger cell.

D) GM-CFC, this colony is made up of medullary system, comprises scavenger cell, granulocyte and megalokaryocyte.

E) BFU-E, this colony is made up of the cell that belongs to erythron, as acidophilic normoblast and stoning red corpuscle.The redness of these cells represents that they contain many oxyphorases.This colony is very big, illustrates that it comes from utmost point primary stem cell.

Obtained identical result (data not shown goes out) with the B-CLL cell.Untreated B cell does not produce hematopoiesis colony (data not shown goes out).Therefore these results prove, have the hemopoietic stem cell of survival in the blood sample that the monoclonal antibody CR3/43 with II class MHC β chain handles, but then do not exist in the blood sample that is untreated.

Long-term cultivation

The long-term self potential of checking hemopoietic stem cell of measuring.In this was cultivated, most of compositions of marrow hemopoiesis were at in-vitro multiplication.The key character of this cultivation is the hematopoiesis that continues, and this is to take place not adding under the situation of somatomedin.In this measured, hematopoiesis definitely depended on the foundation of the stroma cell adhesion layer of derived from bone marrow.Stroma cell is (by many non-hematopoietic cells, as compositions such as inoblast, adipocytes, and comprise that all belong to the cell type of mesenchyme system) survival, self, propagation and differentiation by providing suitable environment (secretion somatomedin and synthetic cell epimatrix) to promote stem cell, thus hematopoiesis support.

In this measures, handle the B cell that obtains from the buffy coat of healthy blood donor with CR3/43mab, cause forming adhesion layer (also obtaining same result) in several hours adding antibody with the B-CLL cell, and increase in time.

Adhesion layer is made up of stroma cell, stroma cell (cover cell, mainly by the cell of inoblast/mesenchyme type forms/be the big cell of refraction of light during with anti-phase bright-field microscope observation) 12 all in longer time in the g and D (these cells show with the tight of hematopoietic cell and contact) of hematopoiesis support cell.Also can see in groups primitive hematopoietic cell (be also referred to as pebbles district/dark cell bunch) in adhesion layer, they are the sources that produce hematopoietic cell for a long time.

Form by the small circle cell that forms hemopoietic focus bunch at the non-adhesion layer at hypothallus top (light cell bunch).This layer contains the more typing ancestors of stem cell and hemopoietic system.Non-adhesion layer can produce MIX-CFC, GM-CFC, M-CFC, BFU-E (measuring judgement with colony) and CFU-F (colony forming unit-inoblast) (when carrying out subclone with the long-term cultivation base).

I. use the RT-PCR of cell of the antibody treatment of HLA-DR β chain

In Ramos (B lymphoma) that handles with the CR3/43mab of CD34, c-kit (part of STEM CELL FACTOR), ε-oxyphorase (embryo's form of oxyphorase) and β Actin muscle and K562 (erythrocyte leucocythemia) cell, be measured to genetic transcription.

Method

Use RNAZOL (CINA BIOTECH) to extract mRNA before and after treatment at CR3/43mab.At room temperature use 4 μ l standard buffer solutions, 2 μ l dNTPs, 1 μ l RNASIN, 1 μ l reverse primer (random hexamer primer) and 1 μ l MMLV ThermoScript II to hatch 5 minutes, make mRNA accept the reverse transcription that six aggressiveness cause.This mixture was further hatched under 38 ℃ 1 hour.Adopt then to be designed for amplification CD34, c-kit, the primer of the sequence of ε-oxyphorase and β Actin muscle carries out PCR to mixture under standard conditions.According to disclosed data, at Randell Institute Kings institute synthetic primer.

The result

The result that obtained shows that after handling with CR3/43mAb, the level of β Actin muscle mRNA does not change, and and all significantly increases of the level of ε-oxyphorase mRNA.The result of CD34 and c-kit has further supported the detailed data of front, and the reverse differentiation of these digital proof bone-marrow-derived lymphocytes produces hemopoietic stem cell.

The result who obtains from ε-oxyphorase even more interesting is because following of ε-oxyphorase normal circumstances is expressed at embryonic cell.Therefore handle with CR3/43mAb and not only produce hemopoietic stem cell, even can also produce more primary undifferentiated cell, as embryonic stem cell.

J. sum up

In brief, equipment of the present invention can be used for being divided into stem cell with noble cells is reverse.The example that experiment in vitro is described has disclosed very interesting and initiative discovery, this discovery is about the individuality generation of T and bone-marrow-derived lymphocyte and grows, they can be used for producing stem cell, thereby the lymph hematopoietic cell that influenced in about several hours in the peripheral blood sample generates.

Monoclonal antibody with I I class antigen β chain homologous region is handled the peripheral blood sample that obtains from B cell chronic lymphocytic leukemia (B-CLL) patient, cause the relative number of single positive (SP) T cell and their progenitor cell significantly to increase, their progenitor cell is two male for thymocyte mark CD4 and CD8 antigen, and these are while coexpressions.Yet these phenomenons always follow bone-marrow-derived lymphocyte relative number purpose significantly to reduce.When handling same blood sample, do not find these results with the monoclonal antibody of II class antigen α chain homologous region or I class antigen homologous region.

As if the monoclonal antibody with HLA-DR β chain homologous region in equipment of the present invention is handled the whole blood that obtains from B cell chronic lymphocytic leukemia (B-CLL) patient, cause the production of T lymphocyte.This incident is with the appearance of the cell of the appearance of two positive cells of coexpression CD4 and CD8 mark, expression CD34 and the CD4 that follows appearance ⁺CD3 ⁺And CD8 ⁺CD3 ⁺Cell number increase to sign.In addition, the immunophenotype that takes place in these cell production processes changes identical with the change in the thymic cell development, and is all the more so when particularly measuring in time.

Find that bone-marrow-derived lymphocyte always loses marks such as CD19, CD21, CD23, IgM and DR, this and CD34 ⁺And CD34 ⁺CD2 ⁺The appearance of cell, CD7 ⁺The increase of cell, CD8 ⁺CD28 ⁺And CD28 ⁺The increase of cell, CD25 ⁺The increase of cell, CD10 ⁺And CD34 ⁺Cell and CD34 ⁺And CD19 ⁺The appearance of cell, CD5 ⁺Cell and the increase of expressing the antigenic cell of low-level CD45 take place together.These changes are owing to handle with the monoclonal antibody of HLA-DR antigen β chain homologous region.

The immunophenotype relevant with this processing changes and reverse differentiation of B cell and typing subsequently (i.e. typing again) unanimity, because the most of white corpuscles in the B-CLL blood samples of patients are bone-marrow-derived lymphocyte.And B-CLL patient's bone-marrow-derived lymphocyte is induced after handling with the monoclonal antibody of endoxan and HLA-DR antigen β chain becomes the T lymphocyte, and they can be bone-marrow-derived lymphocyte hatch the back reverse for a long time with this processing.

Use CD16﹠amp; 56 and CD8 and CD3 group sample that the monoclonal antibody of HLA-DR antigen β chain is handled analyze, express stable the increasing of relative number of the cell of these marks, this is consistent with the result who measures with CD19 and CD3 and groups such as DR and CD3.The increase of discovering the IgG level of the HIV patient's who carries out with turbidimetry and immunoelectrophoresis treated and untreated samples supernatant liquor illustrates that the B cell has passed through the plasma cell phase.The relative number purpose increase of above-mentioned cell is also followed and is expressed CD56﹠amp; Much more very 16 antigenic median sizes, particle cell existing with high measuring.Also instantaneous the observing of cell that other is very big and particle is very many, they are male for CD34, CD4 CD8 are labeled as two positive.Also observed other instantaneous cell, they are bigger, contain particle, and are positive for CD3 and CD19.The CD25 that exists on most of bone-marrow-derived lymphocytes disappears, and replaces by the new T lymphocyte that forms to express, and observing the lymphocytic number of T always increases.

CD28 has appearred behind the whole blood with the monoclonal antibody treatments B-CLL patient of DR antigen β chain homologous region ⁺CD8 ⁺And CD28 ⁺Cell.These discoveries are owing to handled blood with the monoclonal antibody of HLA-DR antigen β chain homologous region.

The T lymphocyte that produces in this way generates also peripheral blood, Cord blood, marrow, the HIV at the healthy blood donor ⁺Observe the enriching section of the patient of various infection such as individual and AIDS patient, the bone-marrow-derived lymphocyte that obtains from healthy blood donor blood sample.In addition, the myelocyte labeled analysis that carries out in two B-CLL patients' that handle with the monoclonal antibody of HLA-DR antigen β chain homologous region sample has shown that the relative number of the cell of expressing myelocyte mark such as CD13 and CD33 increases.These marks and CD56﹠amp; 16 or CD7 antigen coexpression.Yet, in independent cell mass, observed the relative number that has the T lymphocyte marker and do not have the antigenic CD7+ cell of myelocyte.In untreated samples or the sample handled with the monoclonal antibody of I class antigen or HLA-DR antigen α chain homologous region, do not find these specific observationss (seeing

chart

2 and 3).These final results represent that in case excited bone-marrow-derived lymphocyte by HLA-DR antigen β chain, they just can not be returned as T lymphocyte progenitor cell, but may reside in myelocyte and the erythron.

Therefore, digital proof among the present invention, in equipment of the present invention, (i) healthy cell can be converted into the cell surface marker with some other cell line cells and the cell of resemblance from a clone, and (ii) can obtain cell (for example stem cell) from the bone-marrow-derived lymphocyte of differentiation and T lymphocyte with original progenitor cells surface markers and resemblance.

Should be noted that and carried out some experiments with the BCLL cell.The BCLL cell is the ripe bone-marrow-derived lymphocyte that can break up to the final differentiation state of plasma cell.In addition, because chromosome deficiency, they show high-caliber propagation, so have a large amount of bone-marrow-derived lymphocytes in the BCLL blood samples of patients.Compare by the many tumour cells with the prior art description, before being used for method of the present invention, the BCLL cell does not carry out any type of limited reverse differentiation.And they also do not show any feature of undifferentiated cell with the form of genome structure, cell marking or cell appearance.They all are sophisticated bone-marrow-derived lymphocytes.

Therefore, then really not so for the BCLL cell although some malignant cell may have the feature of undifferentiated cell to a certain degree, they are gedanken experiment systems of research bone-marrow-derived lymphocyte.In fact, relevant with these experiments any aspect, BCLL and Daudi cell can not distinguish from normal cell fully.In fact, Martensson et al., 1989, Eu r.J.Immunol.19:1625-1629 (sees P1625 rhs.1 ^5tPara) the verified appropriateness of BCLL cell as model system.

In addition, handle the buffy coat that obtains from the healthy donors blood sample with the CR3/43 monoclonal antibody and make the occurrence rate of CD34 (stem cell labeling) higher, the occurrence rate of CD19 (bone-marrow-derived lymphocyte mark) is lower.

Should be noted that the stem cell that produces with method of the present invention can be the stem cell of any tissue, be not limited to the lymph hemopoietic progenitor cell.

To other modification of the present invention should be tangible for a person skilled in the art.

Those skilled in the art should be readily appreciated that equipment of the present invention is not necessarily limited to use the committed cell's of comprising disclosed herein cell mass and/or reagent, and are applicable to the program that any requirement mixes and hatches with the fluid that contains reagent.

Be incorporated herein above-mentioned all open source literatures as a reference.To various modifications of the present invention and change should be that significantly this does not depart from scope and spirit of the present invention for a person skilled in the art.Although the present invention's contact particular is described, should be appreciated that claimed the present invention should too not be limited to these particular.In fact, to molecular biosciences or those skilled in the relevant art significantly, also should be included in the scope of following claim to implementing the modification that mode of the present invention carries out.

Table 1

Patient's the clinical diagnosis and the laboratory condition of blood sample comprise with after various monoclonal antibodies and other agent treated blood sample and the counting of Ku Erte before (WBC)

Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL
Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL	1	B-CLL	12HR AT 22C	100 ND	86.1 ND	86.1 ND	ANTI-B 50
2	B-CLL	2HR AT 22C 2HR AT 22C	39.1 9.6 39.1 37.7	74.4 63.3 74.4 75.1	29.9 6.1 29.9 28.3	ANTI-B 50 ANTI-B PE 50	1	B-CLL	12HR AT 22C	100 ND	86.1 ND	86.1 ND	ANTI-B 50
2	B-CLL	2HR AT 22C 2HR AT 22C	39.1 9.6 39.1 37.7	74.4 63.3 74.4 75.1	29.9 6.1 29.9 28.3	ANTI-B 50 ANTI-B PE 50	3	B-CLL	6HR AT 22C 6HR AT 22C	39.5 9.3 39.5 37.7	71.9 67.2 71.9 72.5	28.3 6.2 28.3 27.4	ANTI-B 50 ANTI-B PE 50
4	B-CLL	24HR AT 22C 24HR AT 22C	39 9.3 39 36.2	73 66.5 73 70.4	28.4 6.2 28.4 25.5	ANTI-B 50 ANTI-B PE 50	3	B-CLL	6HR AT 22C 6HR AT 22C	39.5 9.3 39.5 37.7	71.9 67.2 71.9 72.5	28.3 6.2 28.3 27.4	ANTI-B 50 ANTI-B PE 50
4	B-CLL	24HR AT 22C 24HR AT 22C	39 9.3 39 36.2	73 66.5 73 70.4	28.4 6.2 28.4 25.5	ANTI-B 50 ANTI-B PE 50	5	B-CLL	2HR AT 22C				ANTI-B 50 ANTI-A 50 ANTI-I 50 ANTI-B ﹠ cell toxicant reagent 25+25

Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL
Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL	6	B-CLL	24HR AT 22C				ANTI-B 50
7	B-CLL	24HR AT 22C	170 128 178 130	95.4 91.1 94.2 90.4	16.9 11.6 16.8 11.9	ANTI-B 10 ANTI-I 10 ANTI-B ﹠ cell toxicant reagent 10+20	6	B-CLL	24HR AT 22C				ANTI-B 50
7	B-CLL	24HR AT 22C	170 128 178 130	95.4 91.1 94.2 90.4	16.9 11.6 16.8 11.9	ANTI-B 10 ANTI-I 10 ANTI-B ﹠ cell toxicant reagent 10+20	8	B-CLL	24HR AT 22C	16 7	81.9 51.2	14 3.0	ANTI-B 20
9	B-CLL	12HR AT 22C	+++ 89.5 +++ +++ 95.4	87 85.1 85.4 89.4 84.9	+++ 76.2 +++ +++	ANTI-B 30 ANTI-I 30 ANTI-4 30 ANTI- I+II+4 10+10+10	8	B-CLL	24HR AT 22C	16 7	81.9 51.2	14 3.0	ANTI-B 20
9	B-CLL	12HR AT 22C	+++ 89.5 +++ +++ 95.4	87 85.1 85.4 89.4 84.9	+++ 76.2 +++ +++	ANTI-B 30 ANTI-I 30 ANTI-4 30 ANTI- I+II+4 10+10+10	10	B-CLL	2HR AT 22C	19.3 ND	86 ND	16.7 ND	ANTI-B 30 ANTI-I 30
92	The outpatient	2HR AT 22C	5.4 ND	74.5 ND	ND	ANTI-B	10	B-CLL	2HR AT 22C	19.3 ND	86 ND	16.7 ND	ANTI-B 30 ANTI-I 30	20
92	The outpatient	2HR AT 22C	5.4 ND	74.5 ND	ND	ANTI-B	87	The outpatient	2HR AT 22C	4.8 ND	59.3 ND	ND	ANTI-B	20	20
91	The outpatient	2HR AT 22C	4.2 ND	54.0 ND	ND	ANTI-B	87	The outpatient	2HR AT 22C	4.8 ND	59.3 ND	ND	ANTI-B	20	20
91	The outpatient	2HR AT 22C	4.2 ND	54.0 ND	ND	ANTI-B	21	The outpatient	2HR AT 22C	3.9 ND	47.4 ND	ND	ANTI-B	20	20

Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL
Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL	34	The outpatient	2HR AT 22C	7.2 ND	20.0 ND	ND	ANTI-B	20
36	The baby	4HR AT 22C	13.4 ND	7.3 ND	ND	ANTI-B	34	The outpatient	2HR AT 22C	7.2 ND	20.0 ND	ND	ANTI-B	20	20
36	The baby	4HR AT 22C	13.4 ND	7.3 ND	ND	ANTI-B	93	The HIV+ baby	4HR AT 22C	5.6 ND	43.4 ND	ND	ANTI-B	20	20
BB/ST	40% immature cell 6 ages in days in the blood	2HR AT 22C 24HR AT 22C	60.5 ND	20.2 ND	12.2 ND	ANTI-B 50 ANTI-A 50 ANTI-AB 25+25	93	The HIV+ baby	4HR AT 22C	5.6 ND	43.4 ND	ND	ANTI-B	20
BB/ST	40% immature cell 6 ages in days in the blood	2HR AT 22C 24HR AT 22C	60.5 ND	20.2 ND	12.2 ND	ANTI-B 50 ANTI-A 50 ANTI-AB 25+25	HIV25	AIDS	2HR AT 22C	7.5 ND	34.8 ND	2.6 ND	ANTI-B 50 ANTI-A 50 ANTI-AB 25+25
43/BD	The B cell lacks	4HR AT 22C				ANTI-B 20 ANTI-I 20 ANTI-4 20	HIV25	AIDS	2HR AT 22C	7.5 ND	34.8 ND	2.6 ND	ANTI-B 50 ANTI-A 50 ANTI-AB 25+25
43/BD	The B cell lacks	4HR AT 22C				ANTI-B 20 ANTI-I 20 ANTI-4 20	OB/BD	The B cell lacks	4HR AT 22C				ANTI-B 20 ANTI-I 20 ANTI-4 20
Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL	OB/BD	The B cell lacks	4HR AT 22C				ANTI-B 20 ANTI-I 20 ANTI-4 20
Patient ID	Diagnosis	Laboratory condition	White corpuscle/L X10-9 B A	% lymphocyte B A	# lymphocyte/L 10X-9 B A	Reagent ML/mL	HIV+	AIDS	6HR AT 22C				ANTI-B 20 ANTI-I
IgA-D	IgA lacks	6HR AT 22C				ANTI-B 20 ANTI-I 20	HIV+	AIDS	6HR AT 22C				ANTI-B 20 ANTI-I

EXPT COND: laboratory condition

B: before

A: afterwards

The monoclonal antibody of ANTI-B:HLA-DR antigen β chain homologous region

The monoclonal antibody of ANTI-A:HLA-DR antigen α chain homologous region

The monoclonal antibody of ANTI-I:I class antigen homologous region

ANTI-AB: ANTI-B of Jia Ruing and ANTI-A together

The antigenic monoclonal antibody of ANTI-4:CD4

ANTI-I+II+4: ANTI-I of Jia Ruing and ANTI-B and ANTI-4 together

Cell toxicant reagent: endoxan

ML/ml: every milliliter of microlitre

L: rise

Table 2

Handle before the blood sample and the patient's of the B-CLL that carries out with the monoclonal antibody of CD19 and CD3 afterwards and other situation immunophenotype analysis with the monoclonal antibody of HLA-DR β chain homologous region

The patient	％CD19+	％CD3+	％CD19+CD 3+	％CD3- CD19-	％ CD19+HG CD3-FC+
The patient	％CD19+	％CD3+	％CD19+CD 3+	％CD3- CD19-	％ CD19+HG CD3-FC+		B A	B A	B A	B A	B A
1	88 40	5 19	1 2	6 26	0 12		B A	B A	B A	B A	B A
1	88 40	5 19	1 2	6 26	0 12	2	73 15	10 33	2 7	15 41	0 5
3	73 11	11 33	2 2	14 52	0 2	2	73 15	10 33	2 7	15 41	0 5
3	73 11	11 33	2 2	14 52	0 2	4	71 13	11 37	2 2	16 47	0 2
5	85 40	5 16	1 1	6 26	3 18	4	71 13	11 37	2 2	16 47	0 2
5	85 40	5 16	1 1	6 26	3 18	6	85 43	5 18	1 1	6 27	3 10
7	90 72	2 4	0 2	7 8	0 14	6	85 43	5 18	1 1	6 27	3 10
7	90 72	2 4	0 2	7 8	0 14	8	62 25	7 13	0 1	29 55	2 6
9	90 85	2 3	0 0	2 1	1 4	8	62 25	7 13	0 1	29 55	2 6
9	90 85	2 3	0 0	2 1	1 4	10	78 50	7 14	0 0	14 26	0 8
92	12 10	38 49	0 1	49 40	0 0	10	78 50	7 14	0 0	14 26	0 8
92	12 10	38 49	0 1	49 40	0 0	91	7 3	35 29	0 1	59 67	0 0
87	5 3	32 38	1 1	63 58	0 0	91	7 3	35 29	0 1	59 67	0 0
87	5 3	32 38	1 1	63 58	0 0	21	1 1	27 29	1 0	71 70	0 0
34	1 1	13 13	0 2	86 84	0 0	21	1 1	27 29	1 0	71 70	0 0
34	1 1	13 13	0 2	86 84	0 0	39	10 6	23 25	0 0	67 69	0 0
93	6 3	26 27	1 1	68 70	0 0	39	10 6	23 25	0 0	67 69	0 0
93	6 3	26 27	1 1	68 70	0 0	BB/ST	1 1	12 13	0 0	87 86	0 0
HIV25	7 2	26 27	0 0	68 67	0 0	BB/ST	1 1	12 13	0 0	87 86	0 0
HIV25	7 2	26 27	0 0	68 67	0 0	43/BD	0 0	40 42	0 1	58 54	0 0
						43/BD	0 0	40 42	0 1	58 54	0 0
						04/BD	0 0	49 41	0 3	43 41	0 0
HIV+	1 1	10 14	0 0	89 87	0 0	04/BD	0 0	49 41	0 3	43 41	0 0
HIV+	1 1	10 14	0 0	89 87	0 0	IgA/D	10 1	21 25	2 3	67 71	0 0

B: A before handling: after the processing

Table 3

Handle before the blood sample and the patient's of the B-CLL that carries out with the monoclonal antibody of CD4 and CD8 afterwards and other situation immunophenotype analysis with the monoclonal antibody of HLA-DR β chain homologous region

The patient	％CD8+	％CD4+	％CD4+CD 8+	％CD4- CD8-	CD4+LO W
The patient	％CD8+	％CD4+	％CD4+CD 8+	％CD4- CD8-	CD4+LO W		B A	B A	B A	B A	B A
1	2.8 16	2.9 11.4	0 3.2	93.1 67.6	0 0		B A	B A	B A	B A	B A
1	2.8 16	2.9 11.4	0 3.2	93.1 67.6	0 0	2	6.2 13.2	9.1 24.3	0 9.4	78.7 46	5.8 6.3
3	7.2 13.1	7.4 23.9	0 8.2	78.8 48.1	6.3 6.6	2	6.2 13.2	9.1 24.3	0 9.4	78.7 46	5.8 6.3
3	7.2 13.1	7.4 23.9	0 8.2	78.8 48.1	6.3 6.6	4	10.1 24.2	7.6 24.9	0.3 2.8	77.5 42	4.6 5
5	2.9 16.2	1.8 7.6	0 2	95 62.3	0 0	4	10.1 24.2	7.6 24.9	0.3 2.8	77.5 42	4.6 5
5	2.9 16.2	1.8 7.6	0 2	95 62.3	0 0	6	ND 12	ND 8.1	ND 1.7	ND 75.7	ND 0
7	1.9 2.6	1.9 2.8	0 0	95.8 94.3	0 0	6	ND 12	ND 8.1	ND 1.7	ND 75.7	ND 0
7	1.9 2.6	1.9 2.8	0 0	95.8 94.3	0 0	8	3.2 7	3.9 6.9	0.1 2	87.3 79.8	4.3 6
9	2.8 2.9	3 3	0 0	94 94.1	0 0	8	3.2 7	3.9 6.9	0.1 2	87.3 79.8	4.3 6
9	2.8 2.9	3 3	0 0	94 94.1	0 0	10	5.7 9.4	4.7 9.1	0.6 0.8	88.7 79.2	0 0
92	21 19	21.6 21	0.8 1.9	50.5 52.5	5.3 4.8	10	5.7 9.4	4.7 9.1	0.6 0.8	88.7 79.2	0 0
92	21 19	21.6 21	0.8 1.9	50.5 52.5	5.3 4.8	91	15.4 18.1	13.6 17.9	6.2 2.6	57 57.3	7.3 3.5
87	16.8 21.8	13.4 20.4	2.9 2.6	59.5 48.9	7 5.6	91	15.4 18.1	13.6 17.9	6.2 2.6	57 57.3	7.3 3.5
87	16.8 21.8	13.4 20.4	2.9 2.6	59.5 48.9	7 5.6	21	16 24.1	9.1 15.2	1 2.6	69.6 53.2	3.7 4.2
34	9.4 11.9	5.7 4.9	2 3.3	67.6 65.3	14.4 14.5	21	16 24.1	9.1 15.2	1 2.6	69.6 53.2	3.7 4.2
34	9.4 11.9	5.7 4.9	2 3.3	67.6 65.3	14.4 14.5	39	12.1 12.6	13.1 14.6	0.4 1.3	62.3 66.7	11.9 4.3
93	18.9 20.3	9.7 10.3	1.8 1.4	65.5 65.9	3.4 1.8	39	12.1 12.6	13.1 14.6	0.4 1.3	62.3 66.7	11.9 4.3
93	18.9 20.3	9.7 10.3	1.8 1.4	65.5 65.9	3.4 1.8	BB/ST	6.3 13	5.7 7.3	2.2 1.1	34.7 70.3	50.3 7.6
HIV25	24.1 24.9	0.8 1.1	1.3 5	70.2 69.3	2.9 3.8	BB/ST	6.3 13	5.7 7.3	2.2 1.1	34.7 70.3	50.3 7.6

Table 4

Handle before the blood sample and the patient's of the B-CLL that carries out with the monoclonal antibody of CD3 and DR afterwards and other situation immunophenotype analysis with the monoclonal antibody of HLA-DR β chain

Table 5

Handle before the blood sample and the patient's of the B-CLL that carries out with the monoclonal antibody of CD16+56 and CD3 afterwards and other situation immunophenotype analysis with the monoclonal antibody of HLA-DR β chain homologous region

Table 6

Handle before the blood sample and the patient's of the B-CLL that carries out with the monoclonal antibody of CD45 and CD14 afterwards and other situation immunophenotype analysis with the monoclonal antibody of HLA-DR β chain

Table 7

Handle before the blood sample and the patient's of the B-CLL that carries out with the monoclonal antibody of CD8 and CD3 afterwards and other situation immunophenotype analysis with the monoclonal antibody of HLA-DR β chain

Table 8

Handle the measured B-CLL patient's immunophenotype analysis in time of monoclonal antibody that blood sample is used CD45 and CD14 afterwards with the monoclonal antibody of PE link coupled HLA-DR β chain

Time	DR+CD45+CD14+r	CD45+L	CD45+H
Time	DR+CD45+CD14+r	CD45+L	CD45+H	2HR	81.7	8.2	8.2
6HR	80.7	8.1	10.6	2HR	81.7	8.2	8.2
6HR	80.7	8.1	10.6	24HR	79	1.1	18.4

Table 9

Handle the measured B-CLL patient's immunophenotype analysis in time of monoclonal antibody that blood sample is used CD19 and CD3 afterwards with the monoclonal antibody of PE link coupled HLA-DR β chain

Time	CD19+DR+r	CD3+	CD3+DR+	CD19-CD3-DR-
Time	CD19+DR+r	CD3+	CD3+DR+	CD19-CD3-DR-	2HR	87.4	10.1	1.8	10.7
6HR	75.5	10.4	3.1	10.7	2HR	87.4	10.1	1.8	10.7
6HR	75.5	10.4	3.1	10.7	24HR	74	11.7	2.9	11

Table 10

Handle the measured B-CLL patient's immunophenotype analysis in time of monoclonal antibody that blood sample is used CD4 and CD8 afterwards with the monoclonal antibody of PE link coupled HLA-DR β chain

Time	CD8+&DR+r	CD4+	CD4+&CD8+&DR +r	CD4+DR+	CD4-CD8-DR-
Time	CD8+&DR+r	CD4+	CD4+&CD8+&DR +r	CD4+DR+	CD4-CD8-DR-	2HR	77.6	6.8	5.4	1.3	8.8
6HR	75.8	6.7	6.4	1.8	9.3	2HR	77.6	6.8	5.4	1.3	8.8
6HR	75.8	6.7	6.4	1.8	9.3	24HR	77	6.4	4.8	1.9	11

Table 11

Handle the measured B-CLL patient's immunophenotype analysis in time of monoclonal antibody that blood sample is used CD3 and DR afterwards with the monoclonal antibody of PE link coupled HLA-DR β chain

Time	DR+	CD3+	CD3+DR+	CD3+DR-
Time	DR+	CD3+	CD3+DR+	CD3+DR-	2HR	75	9.5	4.2	10.9
6HR	74.8	8.8	4.8	10.9	2HR	75	9.5	4.2	10.9
6HR	74.8	8.8	4.8	10.9	24HR	ND	ND	ND	ND

Table 12

Handle blood sample with the monoclonal antibody of PE link coupled HLA-DR β chain and use CD16﹠amp afterwards; 56 and the measured B-CLL patient's immunophenotype analysis in time of monoclonal antibody of CD3

Time	CD56+&16+DR+r	CD3+	CD56+CD16+&CD3 +DR+r	CD56-CD16- &CD16-DR-
Time	CD56+&16+DR+r	CD3+	CD56+CD16+&CD3 +DR+r	CD56-CD16- &CD16-DR-	2HR	82.5	9.5	4.1	3.5
6HR	84.3	7.5	4.1	3.3	2HR	82.5	9.5	4.1	3.5
6HR	84.3	7.5	4.1	3.3	24HR	ND	ND	ND	ND

Table 13

Handle the measured B-CLL patient's immunophenotype analysis in time of monoclonal antibody that blood sample is used CD8 and CD3 afterwards with the monoclonal antibody of PE link coupled HLA-DR β chain

Time	CD8+DR+	CD3+	CD8+CD+3&DR+r	CD8-CD3-DR-
Time	CD8+DR+	CD3+	CD8+CD+3&DR+r	CD8-CD3-DR-	2HR	76.2	6.6	6.7	10.6
6HR	76.5	6.2	6.2	10.3	2HR	76.2	6.6	6.7	10.6

Table 14

In time the monoclonal antibody of the monoclonal antibody with HLA-DR α chain homologous region of Ce Lianging, HLA-DR β chain homologous region, two kinds of monoclonal antibodies together, the monoclonal antibody of the β chain homologous region monoclonal antibody that adds endoxan and I class antigen homologous region handle before and B-CLL patient's immunophenotype analysis afterwards

Before the B=; After the A=; AB=adds after the antibody of β chain; AA=adds after the antibody of α chain; ABC=adds after the antibody and endoxan of α chain or β chain; A1=adds after the antibody of I class.

Table 15

CD8 and CD4

Table 16

CD3 knows DR

Table 17

CD16﹠amp; 56 and CD3

Table 18

CD45 and CD14

Table 19

CD8 and CD28

Table 20

CD34 and CD2

Table 21

The FACs that carries out with the cell that derives from the yellow layer of human blood piece analyzes

		2h analyzes	2h analyzes	24h analyzes	24h analyzes	5d analyzes	5d analyzes
		2h analyzes	2h analyzes	24h analyzes	24h analyzes	5d analyzes	5d analyzes	The CD mark	Initial analysis	Untreated cell	Treated cell	Treated cell	Treated cell	Untreated cell	Treated cell
CD45								The CD mark	Initial analysis	Untreated cell	Treated cell	Treated cell	Treated cell	Untreated cell	Treated cell
CD45								CD38	54.9	55.6	30.1	51.8	52.8	13.0	5.4
CD34	0.6	0.5	11.5	9.7	8.0	0.4	25.1	CD38	54.9	55.6	30.1	51.8	52.8	13.0	5.4
CD34	0.6	0.5	11.5	9.7	8.0	0.4	25.1	CD10	0.2	0.0	0.9	1.9	1.8	0.0	0.2
CD19	13.2	0.0	15.1	11.1	2.3	8.7	0.7	CD10	0.2	0.0	0.9	1.9	1.8	0.0	0.2
CD19	13.2	0.0	15.1	11.1	2.3	8.7	0.7	CD34	1.8	13.1	0.4	13.0	9.1	0.4	25.8
CD3	56.3	62.6	29.7	46.9	69.2	79.8	63.4	CD34	1.8	13.1	0.4	13.0	9.1	0.4	25.8
CD3	56.3	62.6	29.7	46.9	69.2	79.8	63.4	CD117	0.3	0.6	0.0	1.3	0.8	0.2	0.1
CD34	1.8	0.6	7.4	6.4	6.0	0.2	24.6	CD117	0.3	0.6	0.0	1.3	0.8	0.2	0.1
CD34	1.8	0.6	7.4	6.4	6.0	0.2	24.6	CD71	1.5	0.4	0.0	4.8	3.9	0.2	42.9
GLYCA	5.4	2.3	10.1	3.6	2.8	3.8	1.8	CD71	1.5	0.4	0.0	4.8	3.9	0.2	42.9
GLYCA	5.4	2.3	10.1	3.6	2.8	3.8	1.8	CD34	1.3	0.6	14.3	12.5	8.9	0.4	24.3
CD90	18.4	12.6	0.0	9.3	5.0	0.1	0.3	CD34	1.3	0.6	14.3	12.5	8.9	0.4	24.3
CD90	18.4	12.6	0.0	9.3	5.0	0.1	0.3	AC133	0.1	0.0	2.0	0.3	0.4	0.0	0.1
CD34	1.5	0.6	11.9	10.8	7.7	0.0	13.2	AC133	0.1	0.0	2.0	0.3	0.4	0.0	0.1

Value representation is the cell per-cent of presentation markup

Chart 1

The immunophenotype change of measure in time untreated and patient (2, the 3 and 4) blood sample handled with the monoclonal antibody of HLA-DR antigen β chain homologous region

Not having has the FL1 FL2 time

NOTHING001 WITH002 CD45 CD14 2HR

NO001 WE002 CD45 CD14 6HR

001001 002002 CD45 CD14 24HR

NOTHING003 WITH004 CD3 CD19 2HR

NO003 WE004 CD3 CD19 6HR

001003 002004 CD3 CD19 24HR

NOTHING004 WITH005 CD4 CD8 2HR

NO004 WE005 CD4 CD8 6HR

001004 002005 CD4 CD8 24HR

NOTHING005 WITH006 CD3 DR 2HR

NO005 WE006 CD3 DR 6HR

001005 002006 CD3 DR 24HR

NOTHING006 WITH007 CD3 CD56&16 2HR

NO006 WE007 CD3 CD56&16 6HR

001006 002007 CD3 CD56&16 24HR

N003 W004 CD3 CD8 2HR

NO007 WE008 CD3 CD8 6HR

001007 002008 CD3 CD8 24HR

Chart 1A

The immunophenotype change of measure in time untreated and patient (2, the 3 and 4) blood sample handled with the monoclonal antibody of the HLA-DR antigen β chain homologous region that is coupled to PE

The ID FL1 FL2 time

WL003 CD45 CD14 2HR

WEL003 CD45 CD14 6HR

003003 CD45 CD14 24HR

WL005 CD3 CD19 2HR

WEL005 CD3 CD19 6HR

003005 CD3 CD19 24HR

WL006 CD4 CD8 2HR

WEL006 CD4 CD8 6HR

003006 CD4 CD8 24HR

WL007 CD3 DR 2HR

WEL007 CD3 DR 6HR

WL008 CD3 CD65&162 HR

WEL008 CD3 CD56&16 6HR

WL005 CD3 CD8 2HR

WEL009 CD3 CD8 6HR

Chart 2

The immunophenotype change of measure in time untreated and patient (1) blood sample handled with the monoclonal antibody of the monoclonal antibody of monoclonal antibody, this antibody and the endoxan of HLA-DR antigen β chain homologous region, HLA-DR antigen α chain homologous region and I class antigen homologous region

Have or not the FL1 FL2 time

NA001 CD45 CD14 2HR

A2B001：AB CD45 CD14 2HR

A2A：AA CD45 CD14 2HR

DNAA001：ABC CD45 CD14 2HR

A1001：AI CD45 CD14 2HR

NC001 CD3 CD19 2HR

C2B001：AB CD3 CD19 2HR

C2A001：AA CD3 CD19 2HR

DNAC001：ABC CD3 CD19 2HR

C1001：AI CD3 CD19 2HR

A124H001：AI CD3 CD19 24HR

A2B24H001：AB CD3 CD19 24HR

A2A24H001：AA CD3 CD19 24HR

A2BX24H001：ABC CD3 CD19 24HR

ND001 CD4 CD8 2HR

D2B001：AB CD4 CD8 2HR

D2A001：AA CD4 CD8 2HR

DNAD001：ABC CD4 CD8 2HR

D1001：AI CD4 CD8 2HR

D124H001：AI CD4 CD8 24HR

D2BX24H001：ABC CD4 CD8 24HR

D2B001：AB CD4 CD8 24HR

D2A001：AA CD4 CD8 24HR

E1001：AI CD3 DR 2HR

E2B001：AB CD3 DR 2HR

E2A001：AA CD3 DR 2HR

F1001：AI CD3 CD56&16 2HR

F2B001：AB CD3 CD56&16 2HR

F2A001：AA CD3 CD56&16 2HR

G1001：AI CD28 CD8 2HR

G2A001：AA CD28 CD8 2HR

G2B001：AB CD28 CD8 2HR

H1001：AI CD7 CD33&13 2HR

H2A001：AA CD7 CD33&13 2HR

Have or not the FL1 FL2 time

H2B001：AB CD7 CD33&13 2HR

I2A001：AA CD21 CD5 2HR

I2B001：AB CD21 CD5 2HR

J2A001：AA CD34 CD2 2HR

J2B001：AB CD34 CD2 2HR

B2A24H001：AA CD34 CD2 24HR

B2B24H001：AB CD34 CD2 24HR

B2BX24H001： CD34 CD2 24HR

ABC

K2B001：AB CD10 CD25 2HR

K2A001：AA CD10 CD25 2HR

Chart 3

The immunophenotype of patient (8) blood that handle and that handle with the monoclonal antibody of HLA-DR antigen β chain homologous region changes

Have or not the FL1 FL2 time

AN001 CD45 CD14 2HR

A2001 CD45 CD14 2HR

CN001 CD3 CD19 2HR

C2001 CD3 CD19 2HR

DN001 CD4 CD8 2HR

D2001 CD4 CD8 2HR

EN001 CD3 DR 2HR

E2001 CD3 DR 2HR

FN001 CD3 CD56&16 2HR

F2001 CD3 CD56&16 2HR

GN001 CD28 CD8 2HR

G2001 CD28 CD8 2HR

HN001 CD7 CD5 2HR

H2001 CD7 CD5 2HR

IN001 CD13 CD20 2HR

I2001 CD13 CD20 2HR

JN001 CD45RA CD25 2HR

J2001 CD45RA CD25 2HR

KN001 CD57 CD23 2HR

K2001 CD57 CD23 2HR

Chart 4

The immunophenotype of patient (10) blood sample untreated and that handle with the monoclonal antibody of the monoclonal antibody of HLA-DR antigen β chain homologous region and I class antigen homologous region changes

Have or not the FL1 FL2 time

CLL0001 CD45 CD14 2HR

CLL1001 CD45 CD14 2HR

CLL2001 CD45 CD14 2HR

CLL0003 CD3 CD19 2HR

CD3 CD19 2HR

CLL1003 CD3 CD19 2HR

CLL2003 CD3 CD19 2HR

CLL0004 CD4 CD8 2HR

CLL1004 CD4 CD8 2HR

CLL2004 CD4 CD8 2HR

CLL005 CD3 DR 2HR

CLL1005 CD3 DR 2HR

CLL2005 CD3 DR 2HR

CLL0006 CD3 CD56&16 2HR

CLL1006 CD3 CD56&16 2HR

CLL2006 CD3 CD56&16 2HR

Claims

1. relative number destination device that in comprising committed cell's cell mass, forms undifferentiated cell and/or increase undifferentiated cell, this equipment comprises a chamber, the cell mass that will comprise the committed cell is introduced the instrument of described chamber, reverse differentiation instrument is introduced the instrument of described chamber, and the instrument of hatching of under the condition that described reverse differentiation instrument exists, hatching described committed cell, thereby make the reverse undifferentiated cell that is divided into of committed cell, wherein said reverse differentiation instrument is the ratio destructive instrument that causes normal differentiation cell in the cell mass.

2. relative number destination device that in comprising committed cell's cell mass, forms undifferentiated cell and/or increase undifferentiated cell, this equipment comprises a chamber, the cell mass that will comprise the committed cell is introduced the instrument of described chamber, reagent is introduced the instrument of described chamber, and the instrument of hatching of hatching described reagent and described committed cell, thereby make the reverse undifferentiated cell that is divided into of committed cell, wherein said reagent relates to the acceptor of catching, discerning or present of mediation committed cell surface antigen.

3. according to the equipment of claim 1 or 2, wherein said equipment comprises the survey instrument of measuring described cell mass volume.

4. according to the equipment of claim 1 or 2, wherein said equipment comprises the instrument that carries out Cytometric instrument and measure the cell concn of described cell mass.

5. according to the equipment of claim 4, wherein carrying out Cytometric instrument is Coulter-counter.

6. according to the equipment of claim 5, wherein Coulter-counter is microminiaturized Coulter-counter.

7. according to the equipment of claim 1 or 2, wherein said equipment comprises transfers to means of transferring the described chamber with a certain amount of described cell mass from storage vessel.

8. according to the equipment of claim 1 or 2, wherein said equipment comprises transfers to means of transferring the described chamber with the described cell mass of predetermined amount from storage vessel.

9. according to the equipment of claim 2, wherein said equipment comprises the computational tool that is used for calculating the reagent volume that will be added into chamber.

10. according to the equipment of claim 2, wherein said equipment comprises another kind and is used for the agent transfer of the certain volume means of transferring to chamber.

11. according to the equipment of claim 10, wherein said another kind of means of transferring is motor-driven syringe.

12. according to the equipment of claim 2, wherein said equipment comprises another kind and is used for the agent transfer of the volume calculated means of transferring to chamber.

13. according to the equipment of claim 12, wherein said another kind of means of transferring is motor-driven syringe.

14. according to the equipment of claim 1 or 2, wherein said equipment comprises the carbonic acid gas control tool that is used for controlling described chamber gas concentration lwevel.

15. according to the equipment of claim 1 or 2, wherein said equipment comprises the temperature control tool of the temperature that is used for controlling described chamber.

16. according to the equipment of claim 1 or 2, wherein said equipment comprises the mixing tool that is used at chamber mixed cellularity group and reagent.

17. according to the equipment of claim 1 or 2, wherein said equipment comprises the timing tool that is used for the stage of hatching is carried out timing.

18. according to the equipment of claim 1 or 2, wherein said equipment comprises and is used for showing the show tools of hatching remaining time in stage to the user.

19. according to the equipment of claim 1 or 2, wherein said equipment comprises and is used to the prompting instrument of reminding the user to finish in the stage of hatching.

20. according to the equipment of claim 1 or 2, wherein said equipment comprises the harvesting tool that is used for from the chamber harvested cell.

21. according to claim 20 equipment, wherein harvesting tool is gathered in the crops undifferentiated cell from chamber.

22. according to the equipment of claim 1 or 2, wherein said equipment comprises from chamber the cell sample that the comprises undifferentiated cell taking-up instrument to the storage vessel that takes out.

23. according to the equipment of claim 1 or 2, wherein said equipment comprises the sealing tool of sealed storage container, contains the cell mass that comprises undifferentiated cell in the storage vessel.

24. according to the equipment of claim 1 or 2, wherein the committed cell is not a cancer cells.

25. according to the equipment of claim 1 or 2, wherein the committed cell is a noble cells.

26. according to the equipment of claim 1 or 2, wherein the committed cell is the hematopoietic cell of typing.

27. according to the equipment of claim 1 or 2, wherein the committed cell is selected from the CFC-T cell, CFC-B cell, CFC-Eosin cell, CFC-Bas cell, CFC-GM cell, CFC-MEG cell, CFC-E cell, T cell and B cell.

28. according to the equipment of claim 1 or 2, wherein undifferentiated cell is a multipotential stem cell.

29. according to the equipment of claim 1 or 2, wherein undifferentiated cell is the stem cell that is selected from hemopoietic stem cell, neuronal stem cell, epithelial stem cell, mescenchymal stem cell, endothelial stem cell and embryonic stem cell.

30. according to the equipment of claim 1 or 2, wherein undifferentiated cell is characterised in that following one or more cell surface marker symbol: CD34 ⁺, HLA-DR ^-, CD38 ^-, CD117, AC133, CD90 and/or low CD45.

31. according to the equipment of claim 1 or 2, wherein undifferentiated cell is a MHC I class ⁺And/or MHC II class ⁺Cell.

32. according to the equipment of claim 2, wherein this receptor is MHC I class antigen or mhc class ii antigen.

33. equipment according to claim 32, wherein I class antigen is HLA-A acceptor, HLA-B acceptor, HLA-C acceptor, HLA-E acceptor, HLA-F acceptor or HLA-G acceptor, and described II class antigen is HLA-DM acceptor, HLA-DP acceptor, HLA-DQ acceptor or HLA-DR acceptor.

34. according to the equipment of claim 33, wherein this receptor is the HLA-DR acceptor.

35. according to the equipment of claim 2, wherein this receptor comprises the β chain with homologous region.

36. according to the equipment of claim 35, wherein this receptor comprises the homologous region of HLA-DR β chain at least.

37. according to the equipment of claim 2, wherein this reagent is the antibody of acceptor.

38. according to the equipment of claim 37, wherein this reagent is the monoclonal antibody of acceptor.

39. according to the equipment of claim 37 or 38, wherein antibody is selected from monoclonal antibody CR3/43 and monoclonal antibody TAL 1B5.

40. according to the equipment of claim 2, wherein this reagent is regulated MBC genetic expression.

41. according to the equipment of claim 40, wherein this reagent is regulated MHC I class ⁺And/or MHC II class ⁺Express.

42. according to the equipment of claim 1 or 2, the cell mass that wherein comprises the committed cell is the buffy coat blood sample or comes from the buffy coat blood sample.

43. one kind forms in comprising committed cell's cell mass and has cell surface marker symbol CD34 ⁺And/or HLA-DR ^-And/or CD38 ^-And/or the cell of CD117 and/or AC133 and/or CD90 and/or low CD45 and/or increase its relative number destination device, this equipment comprises a chamber, the cell mass that will comprise the committed cell is introduced the instrument of described chamber, the reagent that operability is related to described committed cell is introduced the instrument of described chamber, and operability is hatched the described committed cell's who is related to by described reagent the instrument of hatching in described chamber, thereby make CD 34 ⁺And/or HLA-DR ^-And/or CD38 ^-And/or the relative number of CD117 and/or AC133 and/or CD90 and/or low CD45 cell is owing to described relating to increases.