CN106267419A

CN106267419A - HIV immunologic purging device

Info

Publication number: CN106267419A
Application number: CN201610539277.0A
Authority: CN
Inventors: 翁炳焕; 李兰娟; 朱晓明; 沈敏; 叶光勇
Original assignee: 翁炳焕
Current assignee: Shu Lan (Hangzhou) Hospital Ltd.; Womens Hospital of Zhejiang University School of Medicine
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2017-01-04
Anticipated expiration: 2036-07-01
Also published as: CN106267419B

Abstract

nullA kind of HIV immunologic purging device for medical domain，It is characterized in that preparation can separated plasma and the separator of hemocyte，Not only former macrophage feature had been retained but also can the hybridoma macrophage strain of indeterminate growth with hybridoma technology preparation，Build CD4+T cell strain with exogenous gene transfection and expand for growth stimulant with the peculiar molecular antibody in its surface，The cell strain of preparation is formulated in cells frozen storing liquid，Depurator is formed with container pack prepared by high-biocompatibility material，Cell concentration is made to reach 80%，Sealing，196 DEG C of liquid nitrogen are medium-term and long-term frozen standby，Macrophage strain therein and CD4+T cell strain all play absorption HIV and are used，Made depurator is applied in combination with separator and regulatory process，Blood in extracorporeal circulation is divided into blood plasma and hemocyte by separator，The purified device of blood plasma converges with hemocyte after filtering HIV，Then feed back.

Description

HIV immunologic purging device

Technical field

The present invention relates to preparation and the application of HIV immunologic purging device in medical domain, be mainly used in HIV sufferers blood plasma acquired immune deficiency syndrome (AIDS) The removing of poison, thus reach to treat the purpose of acquired immune deficiency syndrome (AIDS).

Background technology

After HIV enters human body, first by macrophage phagocytic, but HIV quickly changes the acidity at macrophage some position interior Environment, creates the condition of its existence applicable, is not the most killed and the most within it breeds.Because CD4 is the receptor of HIV, So in macrophage the HIV of breeding by its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays the work of bridge With, utilize self hydrophobic interaction mediate retroviral cyst membrane and cell membrane fusion) enter CD4+ cell (cell, mononuclear phagocyte, Dendritic cell etc.), in intracellular rapid propagation, produce 10 every day⁹～10¹⁰Virion, and constantly enter other normal and The intracellular duplication of CD4+ of regeneration, manufactures more virus infected cell, makes peripheral blood CD4+T cell sustaining breakdown, minimizing. Gp120 with the CD4 receptor of HIV be combined can directly activate infected apoptosis, even infected by HIV T cell express Envelope antigen also can start normal T-cell, is indirectly caused the considerable damage of CD4+ cell by the crosslinking of cell surface CD4 molecule, Result causes the severe immune deficiency centered by CD4+T cell defect, and patient mainly shows: periphery lymphocyte reduces, T4/T8 Proportional arrangement, to phytohaemagglutinin and the loss for reaction of some antigen, delayed allergy declines, NK cell, macrophage Reduced activity, the synthesis of the cytokine such as IL2, IFN-γ reduces.CD4+T cell is most important immunocyte, and the infected one Denier loses a large amount of CD4+T cell, and whole immune system will suffer deathblow, the infection of various diseases is all lost to Drag.HIV can also show as hiding for a long time and not showing clinical symptoms after entering host's CD4+ cell, its geneome RNA Reverse transcription becomes double-stranded DNA, enters in host cell core with viral integrase enzyme, and under the effect of intergrase, double-stranded DNA is incorporated into In host cell gene group, integrated viral DNA is referred to as provirus, and the several months of can hiding does not replicates, and causes AIDS Several months is to incubation period for many years.In the incubation period of AIDS, HIV mainly breeds in the macrophage and dendritic cell of lymph node, These cells are internal HIV depots, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, mitogen, Antigen, TNF, IL-2 and lymph element (LT) can excite HIV provirus gene to replicate at the CD4+T Intracellular transcription infected. After a large amount of propagation, inhibition of HIV granule constantly discharges from destroyed infection cell and is free on blood, then enters back into other Cell continues course of infection.

Body can be stimulated after HIV to produce envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody.At HIV Measuring low-level antiviral neutralizing antibody in carrier, AIDS patients serum, wherein AIDS patients level is minimum, HIV Carrier is the highest, and the most protected effect of this antibody is described.But antibody can not with the viruses contact that retains in mononuclear phagocyte, And HIV envelope protein easily occurs antigenic variation, original antibody is ineffective, makes neutralizing antibody can not play due effect. In the latent infection stage, HIV provirus is integrated in host cell gene group, and therefore HIV will not be identified by immune system, Cannot be removed so only relying on autoimmune function.The critically important reason of another one should be to kill according to antibody, clearly Except the mechanism of antigen speculates, after immune antibody is combined with antigen, if immunological effect to be produced, or by activating complement, Mediation ADCC effect dissolves cellular antigen, but HIV is not cellular antigen；Phagocyte is attracted to gulp down by chemotaxis Bite removing antigen, but HIV is protected in phagocyte on the contrary, breeds；Antibody has been combined neutralization with antigen, makes Lose appeal, but HIV antigenic structure is changeable, often makes antibody be difficult to.

In terms of the treating AIDS method having been used for clinic at present, effect is less preferable: (1) hiv reverse transcriptase inhibitor: The permissive cell being only capable of preventing not yet infected by HIV infects, and does not has therapeutical effect to the cell infected, and toxic and side effects is more, Including gland plastochondria toxicity, bone marrow depression, globular anemia, granulocyte and thrombocytopenia, pancreatitis, crossing drug resistant in addition The generation of property, this kind of medicine is not used alone, and mainly does drug combination, and is easily generated drug resistance mutant, causes clinical efficacy to decline Or lost efficacy.(2) hiv protease inhibitor: be easily generated the toxic and side effects such as drug induced hepatic injury, lipid metabolic disorder and drug resistance. (3) hiv integrase inhibitor: integrated with host cell DNA by suppression inhibition of HIV DNA and play a role, inverse with HIV Transcripting enzyme inhibitor and protease inhibitor associating use simultaneously and antiviral are had synergism.(4) suppression inhibition of HIV enters and presses down Preparation: include that blocking gp120 with CD4 is combined, blocks HIV and be combined with accessory receptor, act on gp41 film subunit and effect Block HIV in T lymphocytic cell surface CC-chemokine receptor 5 (CCR5) and enter host cell, but liver and heart are had pair Effect.(5) cytokine therapy: be mainly used in regulatory T-cell dynamic equilibrium.(6) HIV vaccine treatment: due to HIV's Particularity, is not enough to resist HIV such as inherent immunity and targeting destroys immune system, and virus mutation is rapid, causes the most not yet Develop real safely and effectively vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, including antisense technology, RNA bait, RNA interference, intrabody, dominant negative mutant, suicide gene etc., but enter the II clinical trial phase stage Gene therapy almost without.(8) monoclonal antibody passive immunization therapy: reduce by lowering CD4+T cell surface CCR5 The susceptibility of HIV, the progress delaying acquired immune deficiency syndrome (AIDS) and the diffusion of minimizing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10 should For HIV person, there is good toleration and safety, can delay but bounce-back (9) adoptive immunity of virus can not be stoped Cell therapy: virus a large amount of amplification can be caused during external autologous for mass propgation HIV CD4+T cell, increase the CD4+T that virus infects Cell quantity, and feed back CD4+T cell and may increase the place of internal virus replication, cause virus load to rebound, generally Seeing, adoptive immunity cell therapy, without obvious toxic and side effects, does not obtains satisfied therapeutic effect yet.

CD4 molecule is the receptor of HIV, and HIV is susceptible in CD4+T cell, and CD4+T cell is the one of T lymphocyte, average life Generally about 7 days, but some T cell particularly after immortality chemical conversion cell line (strain) can long-term surviving, infinitely expand.State Outer document is reported, simian virus 40 (SV40) can make some human cell that immortalization occurs.Poulin DL, Kung AL and The research such as Sullivan CS shows, the importing of SV40 T antigen gene can accelerate to convert the growth rate of cell, immortalized cells After the most repeatedly passing on, still there is metastable multiplication characteristic and functional status, also can retain being permitted of its germinal cell simultaneously Many phenotypic differentiations.Vascular smooth muscle cell strain is set up in Reilly simian virus large T antigen gene transformation, builds cell model Inhibitory action mechanism with research heparin for vascular smooth muscle.Su etc. utilize the superficial cell strain converted through SV40, build thin The regulating and controlling effect of epithelial cell internal protein synthesis analyzed by born of the same parents' model.The superficial cell strain that Miquel etc. convert with SV40, Cell adhesion as cell model research laminin，LN 5 mediation.Webber etc. are thin with the prostatic epithelium converted through SV40 Born of the same parents' strain studies physiological function and the secretory function of prostate epithelial cell as cell model.Racusen etc. are with through Ad12-SV40 Convert renal cells model and study damage and the disease of proximal convoluted tubule.Hougton etc. convert with SV40 and set up bone marrow matrix Cell strain is as cell model to study under certain condition of culture, and cell has to adipose cell and osteoblastic two-way differentiation Potential, study further osteoporotic mechanism.Foreign study is it is also shown that import exogenous human reverse transcriptase of telomere (hTERT) Cell can be made to keep normal phenotype and differentiating characteristic.HTERT has the most been utilized to be successfully established the immortalized cells of some cell System, substantially keep that chromosome stabilityX, differentiation be normal, contact inhibition, without the growth characteristics relatively normally such as oncogenicity.In oral cavity Medical domain, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes immortal human Gingival Fibroblasts, tooth Pericyte, pulp cells system and Dental Follicle Cells system, cell population doublings number reaches more than 150 times, and cell all shows original biology Characteristic, can express the associated protein of derived cell after inducing culture.The transfection hTERT such as Kitagawa establishes people and becomes cementum Cell line, cell multiplication reaches more than 200 times, and cell differentiation mark such as alkali phosphatase, type i collagen etc. are expressed stable.Because grinding Studying carefully requirements of one's work, almost every kind of disease has respective cell model.As diabetes cell model, cancer cell model, Transgenic cell model, climacteric syndrome cell model, endometrial cell model, epilepsy cell model, electronic cell mould Type, alcoholic dementia cell model, cerebral edema cell model etc..So, CD4+T cell strain can be prepared, after mass propgation, For preparing depurator, with the HIV in CD4+T cell clearance blood plasma.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophilic granulocyte in peripheral blood, and macrophagocyte is blood In mononuclear cell and multiple organ, tissue in macrophage, both constitute mononuclear phagocyte system.Mononuclear cell is by bone Monocyte precursor Development And Differentiation in marrow forms, and accounts for the 3%-8% of blood middle leukocytes sum, its volume relatively lymphocyte Bigger, mononuclear cell the most only stops 12-24 hour, subsequently into connective tissue or organ, reaches maturity and bites carefully for huge Born of the same parents, macrophage is the differentiation of mononuclear phagocyte system camber, ripe cell type, has stronger phagocytic function, trip Walking macrophage and be more than mononuclear cell several times, last a long time, some months of can surviving in the tissue, the macrophage settled down has difference Title, be Kupffer Cell in liver, in brain for microglia, in bone for osteoclast etc., its express Fc receptor, C3b receptor and CDl4, play defense function in inherent immunity, is also the professional antigen presenting cells participating in adaptive immunity. The CD4 molecule of Expression of Macrophages, is the receptor of HIV (human immunodeficiency virus) (HIV), after HIV enters human body, first suffers macrophage Phagocytosis, but HIV quickly changes the sour environment at some position in macrophage, creates condition of its existence applicable, no But it is not killed amount reproduction the most within it to assemble, then HIV is passed to CD4+T cell.So, can be with conventional hybridization Oncocyte technology of preparing, prepares macrophage hybridoma, for preparing the depurator for the treatment of acquired immune deficiency syndrome (AIDS) after a large amount of amplifications, The HIV in blood plasma is removed with the phagocytic function of macrophage.

In a word, various medicines and biological product cannot effectively kill internal HIV (human immunodeficiency virus), and price, and side effect is big, so far There is no the effective ways for the treatment of acquired immune deficiency syndrome (AIDS), it has also become attack the global problem being unable to for a long time.

Summary of the invention

In order to solve to attack for a long time the global problem in the treating AIDS field being unable to, present inventors have proposed the present invention.

The invention aims to provide HIV immunologic purging device；Another object is intended to provide the preparation of HIV immunologic purging device and answer Use method.

The object of the present invention is achieved like this: preparation energy washed corpuscles and the separator of blood plasma, with the side of exogenous gene transfection Method builds CD4+T cell strain and expands as growth stimulant using the peculiar molecular antibody in its surface, both protects with hybridoma technology preparation Stay former macrophage feature the hybridoma macrophage strain of indeterminate growth to expand parallel again, take CD4+T cell strain and macrophage Strain is formulated in cells frozen storing liquid, prepares can prevent cell and fragment thereof from leaching and can be thin with high-biocompatibility material parcel Born of the same parents' strain phagocytosis, the blood plasma immunologic purging device in absorption HIV offer place, CD4+T cell strain therein and macrophage strain all rise and gulp down Biting and adsorb the effect of HIV, prepared depurator is applied in combination with separator and regulatory process, the blood quilt in extracorporeal circulation Separator is divided into blood plasma and hemocyte, converges with hemocyte, then feed back internal after blood plasma purified device absorption HIV.

The technological core of the present invention is made up of plasma separator and depurator, and wherein plasma separator is used for washed corpuscles and blood plasma, Cleanser in depurator is made up of the CD4+T cell strain and macrophage strain being formulated in cells frozen storing liquid, can ultralow temperature for a long time Preserve, become the permissive cell of HIV because the CD4 molecule of CD4+T cell surface is the receptor of HIV, energy when meeting with HIV Absorption HIV, adsorbed HIV are fixed in depurator with CD4+T cell, again because of the natural phagocytosis of hybridoma macrophage Characteristic, can be swallowed when HIV meets therewith, is fixed in depurator the most therewith, so blood is separated in circulating in vitro After device is divided into blood plasma and hemocyte, blood plasma when flowing through depurator HIV therein because being inhaled by CD4+T cell and hybridoma macrophage Attached and remove, the blood plasma after purification and hemocyte feed back after converging, and the present invention is with the method replacement of external removings HIV for a long time Cannot effectively kill the routine internal anti-reverse transcription drug treatment of HIV.

Detailed description of the invention

Fig. 1 is the application schematic diagram of the HIV immunologic purging device proposed according to the present invention.

Fig. 2 is the internal structure schematic diagram of the separator proposed according to the present invention.

Fig. 3 is the internal structure schematic diagram of the depurator proposed according to the present invention.

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump (3) Being connected with plasma separator (4), plasma separator (4) is through clean in parallel with two of blood plasma pump (6) and circulation line (7) Change device (8), depurator (9) is connected, and is connected with circulation line (10), venous line (5) the most successively, venous line (5) The other end be connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 is plasma separator inner chamber, and 3 is the micropore on plasma separator inner chamber tube wall, 4 Being the hemocyte that can not pass through micropore (3), 5 is blood plasma and the chemical analysis that can pass through micropore (3), and 6 is outside plasma separator Chamber, 7 is blood plasma flow export, and 8 is the hemocyte outlet with switchable valve.

In Fig. 3,1 is free HIV, 2, the 4 hybridoma macrophage strains being respectively fixed in depurator (6), CD4+T Cell, is delayed in depurator (6) after 3,5 respectively HIV and hybridoma macrophage strain, CD4+T Cell binding Conjugate, 7 is the most combined and descending HIV.

Below in conjunction with Fig. 1, Fig. 2 and Fig. 3, the embodiment of the HIV immunologic purging device that the present invention proposes is explained in detail.

One, the preparation of acquired immune deficiency syndrome (AIDS) blood purification agent

(1) preparation of hybridoma macrophage strain

1, primary cell source

(1) single core blood cell: refer to lymphocyte and the mononuclear phagocyte separated from blood with density－gradient centrifuga－tion method. Concrete grammar is: the White Blood Cells Concentrate buying Blood Center or the Cord blood preserved for scientific research, takes 2mL specimen, and PBS liquid will After hemodilution 2～3 times, fully mixing, 6mL anticoagulation dropper is slowly superimposed on along tube wall and has added 4mL lymphocyte Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tube of separation liquid；3 it are divided in pipe after Li Xin Layer, upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, upper, in Having the white cloud and mist layer narrow band based on mononuclearcell at bed boundary is PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC inserts in another 50mL centrifuge tube, adds 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons Clear 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandon supernatant, (PBS+0.5% is new to add Buffer Raw Ox blood serum+2mmol/LEDTA, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add microscope on blood counting chamber Cell (PBMC) sum in 4 block plaid of lower counting.

(2) single core histiocyte: provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to and bite from the huge of spleen Cell, its preparation method is: the 1. acquisition of spleen tissue and transhipment: under the approval of reason committee and patient's informed consent, take The spleen specimen tissue of excision, shreds into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C without nectar In envelope bottle, it is transported to rapidly cell culture chamber.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to aseptic behaviour Station, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in-house blood and to ensure the aseptic of tissue.Mechanical grinding Mill spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 liquid.Filter outstanding with 200 mesh stainless steel filtering nets Being mixed with histiocytic RPMI-1640 liquid, filtrate is that spleen tissue cell suspension is (mainly containing erythrocyte, lymphocyte, huge Phagocyte etc.).3. erythrocytic cracking in spleen tissue cell suspension: then centrifugal with the washing of RPMI-1640 liquid (1000r/min, 3min), to remove cell debris, add Tris-NH4Cl effect 5min, splitting erythrocyte, rapidly from The heart (1000r/min, 3min), removes the splitting erythrocyte fragment in supernatant, centrifugal 3 times of PBS washing, RPMI-1640 Centrifugal 1 time of washing, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, now, suspendible Mainly containing spleen tissue macrophage and lymphocyte in liquid.4. the adhere-wall culture of spleen tissue macrophage: by aforementioned suspension As cultivating cell stock solution, Trypan Blue judges vigor and counts, and adjusting cell concentration with RPMI-1640 liquid is (3～5) ×10⁶/ L, will adjust the cell suspension inoculation of concentration in glass culture bottle, and condition of culture is 37 DEG C, 50mL/LCO₂, 100% Humidity, cultivates 2～3h respectively, observes form under phase contrast microscope.The digestion of adherent spleen tissue macrophage: adherent spleen The digestion of tissue macrophages: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, and gained cell hangs Liquid washing is centrifugal (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or Post operation give up The specimen abandoned extracts preparation, such as peritoneal cavity liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa etc..

(3) amniotic fluid, villus cell: attached hospital for obstetrics and gynaecology of Zhejiang University reproduction genetic laboratory is standby.In reason, committee criticizes Accurate treating excess syndrome tests the residue amniotic fluid after diagnosis report, villus cell with under patient's informed consent, selects exponential phase cell to continue to employ.

2, cell is cultivated and the adherent preliminary sorting of macrophage

Cell is cultivated routinely, but according to the difference of cellularity, suitably adjusts incubation time, and condition of culture etc. is the most adherent Single core blood cell (PBMC) or single core histiocyte (macrophage) are placed in containing RPMI-1640 culture medium by method In culture dish, in 37 DEG C, 5%CO₂Cell culture incubator (Themo electro corporation CLASS 100, the U.S.) in Hatch 2h, after mononuclearcell is adherent, inhale abandon upper strata suspension cell (cell beyond macrophage be difficult to adherent and with upper Layer liquid removes), PBs buffer washs 3 times gently, adds a small amount of RPMI-1640 culture medium, scrapes with cell scraper adherent Cell (predominantly macrophage, but also have other attached cells a small amount of).1 000r/min is centrifuged 5min, abandons supernatant.Sheep Water cell, villus cell are cultivated 1～7 day, occur that cell growth clone, cell growth converge rate and reach the logarithm life of 60～80% Long-term cell, with trypsinization, PBS, obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorting

Employing immunomagnetic beads method sorting cd4 cell: 1. main agents and instrument: CD4 immunomagnetic beads (U.S. of Germany sky Ni biology skill Art company limited)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；New-born calf serum (Hyclone Company)；MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany).2. cd4 cell immunological magnetic bead sorting Method: cell suspension is divided equally to two 1.5mLEppendorf pipe, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, The every 80uLBuffer of re-suspended cell contains cell number 10⁷Individual, every 10⁷Individual cell add 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, and hatches 15min at 4～8 DEG C, uses 1mLBuffer washed cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer re-suspended cell, MS detached dowel is placed in the magnetic field of MACS separator, Rinse with 500uLBuffer, by 500uL cell suspension by detached dowel, rinse detached dowel repetitive operation with 500uLBuffer 3 times, collect effluent, containing non-cd4 cell in effluent, in separator, take out detached dowel, pressurize with 1000uLBuffer Rinsing detached dowel, collect effluent, this is that (cell viability detects cd4 cell: takes 15uL cell before and after cell purification respectively and hangs Liquid mixes with equal-volume trypan blue solution, and the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, meter Calculate the percentage ratio of living cells in 200 cells).The cell now sorted is mainly macrophage.

4, CDl4 cell (macrophage) sorting

CDl4 is mononuclear cell and the distinctive surface marker of macrophage, if in theory from single core histiocyte, amniocyte And villus cell sorts, then gained cell is macrophage；If sorted from single core blood cell, then gained cell bag Include mononuclear cell and macrophage；But because the mononuclear cell life-span is short, only survive 1 day in peripheral blood and to can not show a candle to macrophage easy In adherent growth, so the most substantially removing in the cell attachment of the present invention is cultivated, the cell sorted out is essentially macrophage.

Basic skills is analogous to cd4 cell, uses immunomagnetic beads method.1. reagent: people CDl4 immunomagnetic beads test kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead and specific target Cell one mononuclear cell combines: every 1 × 10⁸Magnetic bead and the 800uL buffer of individual PBMC addition 200uL coupling CDl4 antibody (contain Have the bovine serum albumin 2.5mL and 2mol/L EDTA0.5mL of 10%, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube fully Mixing, hatches 15min for 4 DEG C, and centre can slightly shake 1 time.Taking-up centrifuge tube after 15min, every 1 × 10⁷Individual cell adds 1～2mL pre-cooling buffer, 1 000r/min, centrifugal 8min, abandon supernatant, add 0.5mL buffer and blow and beat into slender Born of the same parents' suspension.(B) collect the mononuclear cell of marked by magnetic bead: be placed on MACS magnetic frame by cell separation post, add 1mL buffer Statocyte detached dowel, treats that no liquid is dripped, and is added in people's cell separation post by above-mentioned cell suspension immediately, uses 0.5mL buffer Rinse cell separation post 3 times.After to be rinsed, add 1mL buffer, emigrated cells detached dowel from magnetic frame, use pin Post quickly promotes, and goes out the cell combined with CDl4 antibody one magnetic bead in detached dowel, is the macrophage of CDl4+.

Additionally, following 2 kinds of methods sorting also can be used, including 1. adherent method: be placed in by PBMC and cultivate containing RPMI-1640 In the culture dish of base, in 37 DEG C, containing 5%CO: cell culture incubator (Themo electro corporation CLASS 100, The U.S.) in hatch 2h.After adherent mononuclear cells, inhaling and abandon upper strata suspension cell, PBs buffer washs 3 times gently, adds A small amount of RPMI-1640 culture medium, scrapes attached cell with cell scraper.1 000r/min is centrifuged 5min, abandons supernatant. 2. flow cytometry: CDl4 labelling: take PBMC, with buffer (containing the bovine serum albumin 2.5mL and 2 of 10% Mol/LEDTA 0.5mL) adjust cell density be 1 × 10⁸/ mL, adds CDl4+-FITC antibody in every milliliter of cell suspension 100uL, 4 DEG C of lucifuge labelling 18min, then addition 1mL streaming buffer is to terminate dyeing in centrifuge tube, PBs washs 3 times, Adjusting cell density with the PBS containing 2% mycillin is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation at stream The upper sorting of formula cell instrument (BD FAcsAriaII, the U.S.), according to the fluorescence intensity of CDl4 antibody, the relative size of cell and The relative particle of cell and the complexity of internal structure, collect the cell of CDl4+.

5, prepared by CDl4 hybridoma cell strain (hybridoma macrophage strain)

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire Sanguis Bovis seu Bubali Clearly (FBS) purchase Jinshi City Hao on daytime ocean biological product science and technology responsibility company limited；DMSO (--methyl sulfoxide) it is domestic analytical reagent.

(2) myeloma cell prepares: merges and takes out the myeloma cell (SP2/0) that a pipe is frozen the last week in liquid nitrogen container, vertical I.e. put into hot water and thaw that (applying most is Sp2/0 cell strain, and this cell strain growth and fusion efficiencies are the best, and itself does not secretes Any heavy chain immunoglobulin or light chain, the Seedling height scale of cell is 9 × 10⁵/ ml, the doubling time is usually 10～15h； The actual application relevant to human body considers select homologous cell strain, if Fu Xiang bio tech ltd, Shanghai is to ATCC cell bank The NCI-H929 human myeloma cell strain introduced).Adding appropriate complete culture solution after thawing, 1000r/m is centrifuged 3min；Weight Multiple 1 time.Precipitate is moved in Tissue Culture Flask, add DMEM culture fluid, put CO2 incubator and cultivate, within 3-4 days, carry out once Pass on or amplification culture, in merging first 24 hours, adjust cell state, it is ensured that before merging, cellular morphology is good, it is vigorous to grow.Add Entering appropriate basal medium in centrifuge tube, after beaing mixing gently, 1000r/m is centrifuged 5-10min, repeated washing cell 2 times.

(3) CDl4 cell (macrophage) to be hybridized prepares: the mononuclear phagocyte of present invention sorting is adjusted with basal medium Whole total cell counts to 1 × 10⁸～2 × 10⁸Merge for cell.Expecting blue dyeing phase-contrast microscopy with platform, viable count should It is qualified higher than 80%.

(4) cell merges: by CDl4 cell (mononuclear phagocyte) and myeloma cell with 10: the ratio of 1-5: 1 add from In heart pipe, being mixed evenly, 1000r/m is centrifuged 5min, supernatant discarded, beats gently at the bottom of pipe to cell without granular precipitate, weight Multiple 2 times.Preheating centrifuge tube is rotated gently, by 1000 μ L's of preheating under aseptic condition after taking-up in 37 DEG C of water-baths 50%PEG3000 is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, afterwards by the base of the 25mL of preheating Basal culture medium is also added drop-wise in centrifuge tube along tube wall in 3-5min, rotating centrifugal pipe lightly during adding, then Being statically placed in 37 DEG C of water-bath 10min, 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T culture medium.The most mixed It is inoculated into after even in 96 well culture plates, is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.

(5) there is the mononuclear phagocyte strain screening of phagocytic function: observe cell growth status in 96 well culture plates, 7-10 days The rear hybridoma that only has can grow division, now discards HAT culture medium, changes complete medium.Cell clone aufwuchsplate Long-pending when reaching 1/10 cell hole, go culture supernatant, select to have the culture hole of the hybridoma cell strain that growth conditions is good, micro- The position of labelling cell strain growth, size under mirror, using aseptic rifle head to draw cell clone in the position of mark has completely to new In the culture hole of culture medium, doubling dilution counts hole to below the most successively, 37 DEG C, cultivates about one week in 5%CO2 incubator, Basis of microscopic observation cell growth status, when cell clone covers with to hole floor space more than 1/10, takes cell or culture supernatant inspection Survey hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain phagocytosis antibacterial Function detection: macrophage is mixed with staphylococcus or Candida albicans bacteria suspension Incubation, smear, fixing, serge blue liquid dyes, in oil Microscopic observation phagocytosis situation, counting phagocytosis antibacterial and unopsonized carefully The macrophage number ratio of bacterium, to swallow antibacterial function strong macrophage alternately positive clone strain.

2. hybridoma macrophage strain phagocytosis HIV Function detection: take the blood plasma of acquired immune deficiency syndrome (AIDS) (AIDS) patient that Disease Control and Prevention Center preserves After being mixed with hybridoma macrophage strain, separate cell strain, PBS 3 times, measure the phagocyte strain through cracking and gulp down Biting the function of HIV, with specific reference to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), Shanghai inspires biotechnology limited Company) operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, The p24 antigen of 40pg/ml, 80pg/ml is as comparison, and lowest detectable limit is less than 5pg/ml, measurement range 0～400pg/ml, Range of linearity 0.5pg/ml～80pg/ml, in 15min, 450nm measures absorbance (OD), blank calibration object absorbance Value not higher than 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, as absorbance ＞ 0.12 Time be considered as positive.Concrete test kit description of pressing operates.

3. the strain of hybridoma macrophage produces macrophage cytokines detection: examine with MIF (MIF) ELISA Test agent box (stamen bio tech ltd, Shanghai hundred) by specification operates, and detection range is 0～800pg/ml, and sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, and the positive is the strongest, and negative reaction is nothing Color or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: on ELISA detector, In 450nm (if developing the color with ABTS, then 410nm) place, survey each hole OD value after returning to zero with blank control wells, if more than the moon of regulation Property comparison 2.1 times of OD value, be the positive, specifically press test kit description and operate.MIF be collection cytokine, somatomedin, Hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays central Effect, plays panimmunity function in various infection and active chronic inflammation disease.

According to testing result, select the cell clone in the culture hole with stronger macrophage function and repeat next round dilution Cultivate, repeat 2-3 wheel, take out after detection function-stable, proceed to culture bottle mass propgation.

(6) preservation of hybridoma macrophage strain and recovery: preserve first 12 hours and adjust cell growth state, take one bottle of growth prosperous Containing, the cell that form is good, suitably make cell suspension after digestion, 1000r/min is centrifuged 5min, removes supernatant, flicks Making cell loose at the bottom of pipe, add 4 DEG C of 9 parts of complete culture solutions preserved and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, -70 DEG C of refrigerator overnight, put into cryopreservation tube in liquid nitrogen container after taking-up and save backup.The hot water of about 40 DEG C is got out before recovery, Cryopreservation tube is carefully taken out from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, after defrosting 1000r/min from Heart 5min, opens cryopreservation tube under aseptic condition in superclean bench, and the cell complete culture solution after thawing washed once, so After be centrifuged 5min, supernatant discarded at 1000r/min, in case making amplification culture.

6, prepared by hybridoma macrophage strain treatment cell

The i.e. amplification cultivation of hybridoma macrophage strain.Cultivation is moved into after using complete culture solution the most resuspended above-mentioned cell precipitation In bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Repeatedly pass on amplification cultivation, until required hybridoma cell strain quantity, Every Secondary Culture positive hybridoma cell strain 10 generation, the function of detection macrophage hybridoma cell strain, see whether stable.Continue Continue and in several bottles, carry out extensive industrialization prepare, save backup.

(2) preparation of CD4+T cell strain

1, the source of primary lymphocyte

Have to sow by way of: 1. frozen in the Infectious Diseases Lab Sample Storehouse that scientific research preserves lymphocyte strain is (through inactivation HIV Totivirus is immune but is uninfected by the lymphocyte of HIV)；2. buy the fresh White Blood Cells Concentrate in blood station, then carry out inactivation HIV sense The lymphocyte of dye strain immunity；3. the T lymphocyte series (strain) directly bought from businessman；4. the umbilical cord stranguria with blood preserved for scientific research Bar cell (through inactivation HIV immunity)；The most directly take from the peripheral blood lymphocyte (for self) of HIV-1 the infected, use Histopaque lymphocyte separation medium separation mononuclearcell (PBMC).

2, the preparation of CD4+T cell

1. main agents and instrument: CD4, CD8 immunomagnetic beads (Mei Tian Ni Bioisystech Co., Ltd of Germany)；Isothiocyanic acid is glimmering Light element CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company)；(perseverance letter in Shanghai is raw for lymphocyte separation medium Change reagent company limited)；Ethylenediaminetetraacetic acid (EDTA), (the Shanghai raw work biotechnology service of 0.2% Trypan Blue liquid Company)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (U.S. of Germany sky limited public affairs of Ni biotechnology Department)；EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. the separation of mononuclearcell (PBMC) (density－gradient centrifuga－tion method): the aseptic 20mL blood sample that takes, and heparin sodium (500IU/mL) 2mL anticoagulant；PBS liquid is dilute by blood Release 2～3 times, fully after mixing, 6mL anticoagulant venous blood dropper is slowly superimposed on along tube wall and has added 4mL lymphocyte and divide Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tube of chaotropic；3 layers it are divided in pipe after Li Xin, Upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, at stratum boundary upper, middle Having the white cloud and mist layer narrow band based on mononuclearcell at face is PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC Insert in another 50mL centrifuge tube, add 5 times and be centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandon supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandon supernatant, (PBS+0.5% is newborn to add Buffer Ox blood serum+2mmol/LEDTA, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add on blood counting chamber under microscope Count cell (PBMC) sum in 4 block plaid.3. CD4+T cell and CD8+T cell is isolated and purified: PBMC cell Suspension is divided equally to two 1.5mLEppendorf pipes, and centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, re-suspended cell is every 80uLBuffer contains cell number 10⁷Individual, every 10⁷Individual cell adds 20uLCD4MicroBeads or CD8MicroBeads, fully Mixing, hatches 15min at 4～8 DEG C, uses 1mLBuffer washed cell, centrifugal (300r/min, 20 DEG C) 10min, discards Supernatant 500uLBuffer re-suspended cell, is placed on MS detached dowel in the magnetic field of MACS separator, floats with 500uLBuffer Wash, by 500uL cell suspension by detached dowel, rinse detached dowel repetitive operation 3 times with 500uLBuffer, collect effluent, Containing non-CD4+T lymphocyte or non-CD8+T lymphocyte in effluent, in separator, take out detached dowel, use 1000uLBuffer Pressure flush detached dowel, collects effluent, and this is CD4+T lymphocyte or (the cell viability detection: thin of CD8+T lymphocyte Born of the same parents take 15uL cell suspension the most respectively and mix with equal-volume trypan blue solution, and the not colored shinny person of basis of microscopic observation is Living cells, the coloring person of swelling is dead cell, calculates the percentage ratio of living cells in 200 cells).

3, amplification in vitro CD4+T cell

Having document to report the stimulant utilizing the monoclonal antibody of T cell surface C D3 molecule to grow as cell, mass propgation ends After growing the T cell that patient separates, feed back as self therapeutic cells.But HIV is numerous also with the cultivation of HIV cell Grow and at endogenous multiplication, the feedback of increment T cell result also in the feedback of increment HIV.The present invention is with SV40 and/or hTERT Immortalization CD4+T cell, and with CD3 monoclonal antibody for cell growth stimulant, a large amount of amplification CD4+T cells.

With the method that CD3 monoclonal antibody is cell growth stimulant it is: by anti-CD49d McAb, (CD4+T cell contains CD3 simultaneously Molecule) it is coated on culture plate stimulation mononuclearcell (lymphocyte) growth, referred to as anti-cd 3 antibodies is coated method, can obtain Well expanding effect, the lymphocyte that should expand in this way has been used for the second stage of clinical treatment of tumor and achieves certain Curative effect.Foreign literature report [Shimizu etc.] has also cultivated the lymphocyte of 5 example full-blown AIDS patients by the method, cultivates 4 It is achieved with the amplification of 1000 times week, and in the cell mass of amplification, CD4+/CD8+T all can expand that (CD4+T cell is brighter in a large number Aobvious).Another kind is the dual anti-cross-linking method of AntiCD3 McAb/CD28, will dual anti-being crosslinking on taste pearl of AntiCD3 McAb/CD28 cultivate as stimulant HIV person's PERIPHERAL BLOOD MONONUCLEAR CELL (lymphocyte), can expand substantial amounts of CD4+T cell, and the CD4+T expanded is thin Born of the same parents have the ability of antagonism HIV, and in its incubation, virus is also below detection level, finds that this may be with CD28 afterwards Providing secondary signal, it is relevant with chemotactic factor that selective induction secretes substantial amounts of Th1 cytokine, with the method amplification CD4+T cell have been used for HIV person clinical treatment feed back, devoid of risk but effect is general.

With the method for hTERT immortalization CD4+T cell it is: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo-hTERT With carrier pLXSNneo, connect hTERT and the pLXSNneo enzyme separated through PCR amplification, gel electrophoresis with Ligation Mix Cut product, build pLXSNneo-hTERT recon, convert DH5a competent cell blue or green with amplification, purification picking resistant to ammonia benzyl Mycin bacterium colony extracting plasmid, imports with lipofection and passes on the T lymphocyte in logarithmic growth in vitro, makes recon with thin The DNA of born of the same parents integrates, and the clone of positive recombinant that amplification culture screen through G418, screen cellular morphology, growth curve, The test of karyotype, nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemistry dye Color, cell generation cycle and apoptosis rate meet immortalized cells characteristic person same or like with primary cell as hTERT The CD4+T cell of immortalization.

With the method for SV40 immortalization CD4+T cell it is: connect simultaneously through BamHI enzyme action with T4 DNA ligase PcDNA3.1 (-) DNA with PCR amplification, the SV40LTag DNA that separates of agarose gel electrophoresis, structure SV40LTag-pcDNA3.1 (-) recombiant plasmid, convert DH5a competent escherichia coli cell with amplification, purification picking resistant to ammonia benzyl The bacterium colony extracting plasmid of penicillin, imports the T lymphocyte of In vitro culture, makes the DNA of recon and cell with lipofection Integrating, with the cell containing positive recombinant of G418 screening, pass on, amplification culture, screening cellular morphology, cell grow SV40 big T gene test in the test of curve, karyotype, nude mice tumorigenesis, transfectional cell DNA, mrna expression product are surveyed Fixed and determined dna sequence result meets immortalized cells characteristic person same or like with primary cell as SV40 immortalization CD4+T cell.

1. with the concrete grammar of hTERT immortalization CD4+T cell

(I) extraction of hTERT: (i) enzyme action pClneo-hTERT:hTERT is positioned at the EcoRI of plasmid pClneo-hTERT And between SalI site, pLXSNneo vector multiple cloning site (MCS) restriction enzyme site Han EcoRI Yu XhoI.Commercially available purchase PCIneo-hTERT plasmid, is dissolved in appropriate ultra-clean H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, Add restricted enzyme EcoR I and each 0.5ul of Xho I, 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, add 5uL Electrophoresis sample loading buffer (also can be by adding 0.5mol/L EDTA) terminates reaction, routinely after PCR method amplification hTERT, Collect amplified matter in case electrophoresis.(ii) hTERT electrophoresis: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, Pour on the gel casting platform sealed, plug sample comb, after gelling is solid, from glue platform, removes envelope band, extracts comb, Putting in the electrophoresis tank added with enough electrophoretic buffers, buffer exceeds gel surface about 1mm, slow with appropriate 10 × sample-adding Rush liquid and prepare hTERT enzyme action sample, then with pipettor, sample is added in sample well, and do suitable standard control thing simultaneously, Connect electrode, make hTERT face south Ghandler motion move, then under the voltage of 1-10V/cm (80V) gel electrophoresis to being sufficiently separated hTERT During the distance of fragment (30min), close power supply.(iii) hTERT purification and recovery: separate hTERT band from agarose: Gel-tape containing target hTERT fragment is cut in loading bag filter under long wave ultraviolet light source, in bag filter, add 2ml Electrophoretic buffer, is allowed to submergence gel, and empties steam bubble, and bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), Addition appropriate amount of buffer solution, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treat hTERT under uviol lamp All removing gel, change direction of an electric field and continue energising 1 minute, from bag filter, sucking-off buffer is in 1-5ml Eppendorf Guan Zhong, adds 1.5 times of volume n-butyl alcohol, and EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, is just sucking upper strata Butanol solution, so repeats secondary, adds equal-volume phenol chloroform (2) and extract 2 times in the solution of lower floor hTERT, on Proceed to clearly in another Eppendorf pipe add 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C overnight, 12000g, at 4 DEG C centrifugal 10 minutes, obtains hTERT precipitation, abandons supernatant, abandons dry ethanol after adding 70% washing with alcohol 2 times, Add 50 μ l TE and dissolve hTERT.Additionally, can also be used with low melting-point agarose gel method, hTERT filter membrane inserted sheet method etc. by purpose HTERT fragment separates from gel, is purified.

(II) connection of hTERT Yu pLXSNneo carrier: take the hTERT composition (0.1-5 μ g) of the 9 above-mentioned purification of μ l, 1 μ l10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C of incubation 24h, build pLXSNneo-hTERT recon.

(III) pLXSNneo-hTERT recon purification, expand, identify: the preparation of (i) E. coli competent: Its basic skills is ice-cold CaCl₂Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses CaCl₂Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to mostly Number coli strains, operating process is summarized as follows: from 37 DEG C cultivate 16～20h fresh plate one single bacterium colony of picking (as Bacillus coli DH 5 2), or 16～20h overnight culture that 1ml is fresh, forward to a 1L containing 100mlLB culture medium or In 500ml flask, cultivate about 2～3h (rotary shakers 200～300r/min), every 20～30min in 37 DEG C of violent shakings Measure OD600 value ≈ 0.4, aseptically antibacterial is transferred to one, in ice-cold 50ml polypropylene centrifuge tube, Place 10～20min on ice, in 4 DEG C with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) with 4000r/min Centrifugal 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, with The ice-cold 0.1mMCaCl of 10ml₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or with its phase The rotary head answered) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the most residual The trace culture fluid stayed flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, now, Cell can be distributed into rapidly aliquot, freeze in liquid nitrogen ,-70 DEG C of storages are standby.(ii) with competence escherichia coli purification, Amplification pLXSNneo-hTERT recon: take 200 μ l transfers from every kind of competent cell suspension with the sterile pipette tip of cooling In aseptic microcentrifugal tube, often pipe adds DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng), revolves gently Turn mix content, placement 30min in ice, centrifuge tube be put into pre-heating to the test tube rack in the circulator bath of 40 DEG C, Placing 90s～2min, do not shake test tube, quickly transfer in ice bath by pipe, make cell cooling 1～2min, every centrifuge tube adds 800 μ lSOC culture medium, are warmed to 37 DEG C with water-bath by culture medium, are then transferred to by pipe on 37 DEG C of shaking tables, incubation 45min Make bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, by proper volume (every 90mm flat board up to 200 μ l) competent cell that converted transfers in the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, and will be flat Plate is placed in room temperature and is absorbed to liquid, is inverted plate, in 37 DEG C of cultivations, may occur in which bacterium colony after 12～16h.(iii) screening, Amplification recon: select single colony inoculation in LB culture medium aseptic for 5mL or abundant cultivation with sterile toothpick or disinfection inoculation pin In base (such as super broth or TB super broth culture medium), after overnight incubation, it is then added to 500mL culture medium Han LB and (contains Have suitable antibiotic) 2L flask in, cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4, for improving yield, should use table Area is relatively big and the flask of band deflection plate is to increase venting quality as far as possible, and shaking speed should be greater than 400r/min), in 4 DEG C, 6000g Centrifugal 10min, by the 4mL resuspended precipitation of GTL solution, and transfers in the high speed centrifugation pipe of a volume >=20mL that (antibacterial is sunk Shallow lake can preserve at-20 DEG C or-70 DEG C of indefinite duration), add the GTE solution containing 25mg/mL lysozyme that 1mL newly joins, resuspended Precipitation, places 10min in room temperature, adds 10mL and newly joins NaOH/SDS solution, and mixes until liquid becomes equal gently One, limpid and thickness, places 10min on ice, adds 7.5mL acetic acid solution, is gently mixed until under viscosity with suction pipe Dropping and form big precipitation, placing 10min on ice, in 4 DEG C, 20 000g are centrifuged 10min, are poured into by supernatant another gently In one clean centrifuge tube, if there being visible drift that the isopropanol of 0.6 times of volume by several layers of filtered through gauze, can be added, Reverse mixing, room temperature places 5～10min, and in room temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs gently Precipitation, the ofest short duration the most centrifugal, suck ethanol, vacuum drying (precipitation can be 4 DEG C of long-term preservations).(iv) restructuring matter The qualification of grain and amplification: the single bacterium colony on picking plate, be inoculated in 3ml and contain in 100ug/ml ampicillin LB culture medium, 37 DEG C, in 250r/min shaking table cultivate, after 14h collect culture, 4 DEG C, 10000r/min be centrifuged 5min, by test kit Description is extracted and purification of Recombinant plasmid in a small amount；With EcoRI and HindIII double digestion recombiant plasmid reaction system: in restricted Cut each 0.5ul of enzyme, 10 × buffer 2ul, recombiant plasmid 10ul, adding water complements to 20ul, 37 DEG C of enzyme action 1h.Digestion products Carrying out 0.8% sepharose electrophoresis, time 30min under 80V voltage conditions, gel imaging system is taken pictures；Measure restructuring routinely The sequence of plasmid；Recombiant plasmid is after enzyme action, order-checking are identified accurately, by the microbionation containing this plasmid to LB culture fluid, Amplification cultivation, carries out heavy dose of plasmid extraction purification by heavy dose of plasmid extraction test kit description, and ultraviolet spectrophotometer measures After plasmid concentration and purity standby.

(IV) CD4+T cell: from the present invention " preparation of (2) CD4+T cell " sampling preparation.

(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5～10nmol/L insulins, the RPMI of 20% hyclone In 1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, one As be inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer: 15% hyclone (FBS)；1% penicillin and Streptomycin；PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, Centrifugal, remove supernatant, standby.

(VI) pLXSNneo-hTERT recon imports T lymphocyte and amplification culture: make in 1.5ml microcentrifugal tube Standby following solutions: pipe A, is dissolved in pLXSNneo-hTERT recon in 100 μ l serum-free mediums；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, and left at room temperature 45min, by nothing Serum free culture system liquid washs above-mentioned T lymphocyte 2 times.1ml is added in Lipofectamine-pLXSNneo-hTERT mixture Serum-free medium, mixes gently, drops in above-mentioned T lymphocyte, and (hyclone is dense to add 1ml serum-free medium Degree is 20ml/L), at CO₂Incubator cultivates 10h, and sucking-off transfection liquid, (hyclone concentration is to add 4ml complete culture solution 20%), continuing to cultivate 20h, discard culture fluid, changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days Select after living cells makees amplification culture, then strengthen G418 concentration to 800mg L^-1, by can in the G418 environment of high concentration steady The cell of fixed growth proceeds amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, occur poly- Collection phenomenon.If cell increases slow, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out Amount changes liquid.Transfer in 75ml culture bottle when total amount reaches 14ml, every 2-3 week adds 5-10ml fresh culture.Cell Cultivating to 9-10 week (the about the 75th generation), still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, Dead cell (judges the increase situation of cell quantity less than 10% by reading the scale of culture vessel；Pass through trypan blue staining Differentiate dead cell and living cells.Because normal living cells, after birth structural integrity, it is possible to repel, make trypan blue to enter Intracellular；And the cell of loss of activity, the permeability of after birth increases, and can be dyed blueness by trypan blue, can determine whether as cell the most dead Die.Method is to draw weekly a certain amount of suspension culture, mixes rearmounted room temperature 5～10 minutes with Trypan Blue agent, so After make cell sheet, 1000 total cellular score of counting under the microscope, calculate the dead cell of coloring and non-staining living cells Percentage ratio).Hereafter along with increase and the prolongation of incubation time of cultivation algebraically, the increase of cell quantity is slack-off, dead cell more comes The most, until cell is not further added by, even dissolve, reduce, all dead.When total amount reaches 45ml, put 50ml and be centrifuged Guan Zhong, 10 minutes, after abandoning supernatant, adds 3ml freezing media (5% DMSO (dimethyl by centrifugal 1500 turns Sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, Put-20 DEG C of 2h, then put-70 DEG C of 2h, In liquid nitrogen container).

(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: take growth preferably transfection Cell, makes cell suspension, through counting, takes 1.4 × 10 respectively⁴Cell is inoculated in 30 low sugar DMEM culture medium Han 15FBS Culture bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation, until cell quantity is decreased obviously, is cultivated 3 days After gave no count every 2 days cell change liquid, use same method to observe transfectional cell and train at hepato ZYME-SFM serum-free Support the growing state in base.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale Make after curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof "；(iii) Check chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", then Illustrate that this cell line does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA group in flow cytometry analysis cell line Body, if it did not, there is not tumor feature in explanation cell line yet).Chromosome karyotype analysis method is: add by 5mL culture fluid Enter preheating 250ug/ml Colchicine 100ul, mix rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, Fixing, film-making, G show band post analysis karyotype；(iv) Flow cytometry: detection the 19th continuous cell line synthesizes, The cell proportion of division, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is that hTERT integrates, expresses Result.(vii) determined dna sequence: sequenator detection routinely, shows hTERT gene order.(v) transfectional cell DNA Middle hTERT detects: as with Immunohistochemical detection, dye in the nucleus of hTERT transfection visible a large amount of brown particles, table Bright hTERT has been integrated into intracellular；(vi) mrna expression product measures: take the pcr amplification product of 100 μ l systems, uses Gel reclaims test kit (Takara, Japan) and reclaims product, takes 2 μ l DNA solutions and dilutes 100 times, surveys concentration, remaining DNA and each 10 μ l of upstream and downstream primer check order.

(VIII) hTERT mediates CD4+T cell bank: screen and continue to pass on, amplification culture meets immortality after above-mentioned qualification Change cell characteristics the cell same or like with primary cell, take the different generations that growth conditions is good, be in exponential phase Cell, be performing centrifugal separation on (1200r/min, 6min), with the frozen stock solution 0.5～1ml re-suspended cell containing dimethyl sulfoxide, Cell density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter -196 DEG C of liquid nitrogen cryopreservations, the immortalization CD4+T cell bank building biological characteristics stable in this way is standby.

2. with the concrete grammar of SV40 immortalization CD4+T cell

(I) extraction of SV40 large T antigen DNA: (i) SV40DNA enzyme action: contain large T antigen gene from commercially available purchase SV40 freeze dried powder or SV40 plasmid, be dissolved in appropriate H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds restricted enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, adds 5uL electrophoresis sample loading buffer (also can by add 0.5mol/L EDTA) terminates reaction in case electrophoresis.(ii) SV40DNA electricity Swimming: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours on the gel casting platform sealed, inserts Upper sample comb, removes envelope band after gelling is solid from glue platform, extracts comb, put into the electrophoresis added with enough electrophoretic buffers In groove, buffer exceeds gel surface about 1mm, prepares DNA sample with 10 appropriate × sample loading buffer, then uses pipettor Sample is added in sample well, and does suitable DNA molecular amount standard control thing simultaneously, connect electrode, make DNA face south Ghandler motion Dynamic, under the voltage of 1-10V/cm gel, electrophoresis is to when being sufficiently separated the distance of DNA fragmentation, closes power supply.(iii) from fine jade Lipolysaccharide separates about 2600bp SV40 large T antigen DNA: under 300-360nm long wave ultraviolet light source, (use long wave ultraviolet Light source is to prevent DNA damage) gel-tape containing target DNA fragments is cut in loading bag filter, in bag filter, add 2ml Electrophoretic buffer, is allowed to submergence gel, and empties steam bubble, and bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), Addition appropriate amount of buffer solution, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treat DNA under uviol lamp All removing gel, change direction of an electric field and continue energising 1 minute, from bag filter, sucking-off buffer is in 1-5ml Eppendorf Guan Zhong, adds 1.5 times of volume n-butyl alcohol, and EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, is just sucking upper strata Butanol solution, so repeats secondary, adds equal-volume phenol chloroform (2) and extract 2 times in the solution of lower floor speech DNA, on Proceed to clearly in another Eppendorf pipe add 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C overnight, 12000g, at 4 DEG C centrifugal 10 minutes, obtains DNA precipitation, abandons supernatant, abandons dry ethanol, add after adding 70% washing with alcohol 2 times Enter 50 μ l TE dissolving DNAs.Additionally, can also be used with low melting-point agarose gel method, DNA filter membrane inserted sheet method etc. by target DNA sheet Section separates from gel, is purified.

(II) SV40 large T antigen DNA and the connection of pcDNA3.1 genophore: take 9 μ l above-mentioned DNA composition (0.1- 5 μ g), 10 μ l 2 × connect buffer, 1 μ l 10mmol/L ATP, T4 DNA ligase (20～500 sticky ends Unit) or e. coli dna ligase, the mixing of peDNA3.1 empty carrier, 15 DEG C of incubation 24h, it is built into SV40T/pcDNA3.1 Recon.

(III) SV40T/pcDNA3.1 recon amplification, separate and identify: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses CaCl₂Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to mostly Number coli strains, operating process is summarized as follows: from 37 DEG C cultivate 16～20h fresh plate one single bacterium colony of picking (as Bacillus coli DH 5 2), or 16～20h overnight culture that 1ml is fresh, forward to a 1L containing 100mlLB culture medium or In 500ml flask, cultivate about 2～3h (rotary shakers 200～300r/min), every 20～30min in 37 DEG C of violent shakings Measure OD600 value ≈ 0.4, aseptically antibacterial is transferred to one, in ice-cold 50ml polypropylene centrifuge tube, Place 10～20min on ice, in 4 DEG C with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) with 4000r/min Centrifugal 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, with The ice-cold 0.1mMCaCl of 10ml₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or with its phase The rotary head answered) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the most residual The trace culture fluid stayed flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, now, Cell can be distributed into rapidly aliquot, freeze in liquid nitrogen ,-70 DEG C of storages are standby, with the sterile pipette tip of cooling from every kind of competence Cell suspension respectively takes 200 μ l and transfers in aseptic microcentrifugal tube, DNA or coupled reaction mixture (body should be added in often pipe Long-pending≤10 μ l, DNA≤50ng), rotate gently to mix content, ice is placed 30min, centrifuge tube is put into pre-heating On test tube rack in the circulator baths of 40 DEG C, place 90s～2min, do not shake test tube, quickly pipe is transferred in ice bath, Making cell cooling 1～2min, every centrifuge tube adds 800 μ lSOC culture medium, with water-bath, culture medium is warmed to 37 DEG C, then will Pipe is transferred on 37 DEG C of shaking tables, and incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, The competent cell that proper volume (each 90mm flat board is up to 200 μ l) has converted is transferred to containing 200mmol/LMgS04 and In the SOB culture medium of corresponding antibiotic, flat board is placed in room temperature and is absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, 12～ Bacterium colony is may occur in which after 16h.(ii) recon screening, expand and extract: select single bacterium with sterile toothpick or disinfection inoculation pin Fall to being inoculated in LB culture medium aseptic for 5mL or rich medium (such as super broth or TB super broth culture medium), cultivate After overnight, it is then added in the 500mL 2L flask containing LB culture medium (containing suitable antibiotic), cultivates to full then at 37 DEG C With state (OD₆₀₀≈ 4, for improving yield, should use surface area relatively big and the flask of band deflection plate is to increase venting quality as far as possible, shake Shake speed and should be greater than 400r/min), in 4 DEG C, 6000g is centrifuged 10min, by the 4mL resuspended precipitation of GTL solution, and shifts In the high speed centrifugation pipe of a volume >=20mL (bacterial precipitation can preserve at-20 DEG C or-70 DEG C of indefinite duration), add 1mL The GTE solution containing 25mg/mL lysozyme newly joined, resuspended precipitation, place 10min in room temperature, add 10mL and newly join NaOH/sDS Solution, and mixing, until liquid becomes homogeneous, limpid and thickness, places 10min on ice gently, adds 7.5mL acetic acid Solution, is gently mixed with suction pipe until viscosity declines and formed big precipitation, places 10min on ice, in 4 DEG C, and 20 000 G is centrifuged 10min, is poured into gently by supernatant in another clean centrifuge tube, if there being the visible drift can be with several layers of gauze Filtering, add the isopropanol of 0.6 times of volume, reverse mixing, room temperature places 5～10min, and in room temperature, 1 500g is centrifuged 10 Min, adds 2mL 70% ethanol and washs precipitation gently, the ofest short duration the most centrifugal, sucks ethanol, and is vacuum dried that (precipitation can 4 DEG C of long-term preservations).(iii) qualification of recon: the above-mentioned DNA extracted from competence escherichia coli is (containing recon SV40T/pcDNA3.1), ibid method restricted enzyme BamH I carries out enzyme action, and 10g/L agarose gel electrophoresis is identified, Obtaining 2 bands of size about 2600bp and 5600bp, the former meets the size of SV40T fragment in GenBank.

(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5～10nmol/L insulins, the RPMI of 20% hyclone In 1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, one As be inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer；15% hyclone (FBS)；1% penicillin and Streptomycin；PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, Centrifugal, remove supernatant, standby.

(VI) importing of SV40T/pcDNA3.1 and amplification culture: prepare following solutions in 1.5ml microcentrifugal tube: pipe A, SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free mediums (hyclone concentration is 20ml/L)；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, the underlying 45min of room temperature.Use depletion of blood Clear culture fluid washs above-mentioned T lymphocyte 2 times.1ml is added in Lipofectamine-SV40T/pcDNA3.1 mixture Serum-free medium, mixes gently, then drops to, in above-mentioned T lymphocyte, be subsequently adding 1ml serum-free medium (tire cattle Serum-concentration is 20ml/L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone Concentration is 20%), continue to cultivate 20h, discard culture fluid, changing concentration is 400mg L^-1G418 culture fluid continue cultivate, Select after living cells makees amplification culture after 8 days, then strengthen G418 concentration to 800mg L^-1, by can be at the G418 environment of high concentration In the cell of stable growth proceed amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and goes out Existing clustering phenomena.If cell increases slow, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, enters Row equivalent oil changing.Transfer in 75ml culture bottle when total amount reaches 14ml, every 2-3 week adds 5-10ml fresh culture. Cell cultivates about 6-8 week (the about the 55th generation), and still in logarithmic growth, i.e. incubation time and cell is accelerated in multiplication relation, Dead cell (judges the increase situation of cell quantity less than 10% by reading the scale of culture vessel；Pass through trypan blue staining Differentiate dead cell and living cells.Because normal living cells, after birth structural integrity, it is possible to repel, make trypan blue to enter Intracellular；And the cell of loss of activity, the permeability of after birth increases, and can be dyed blueness by trypan blue, can determine whether as cell the most dead Die.Method is to draw weekly a certain amount of suspension culture, mixes rearmounted room temperature 5～10 minutes with Trypan Blue agent, so After make cell sheet, 1000 total cellular score of counting under the microscope, calculate the dead cell of coloring and non-staining living cells Percentage ratio).Hereafter along with increase and the prolongation of incubation time of cultivation algebraically, the increase of cell quantity is slack-off, dead cell more comes The most, until cell is not further added by, even dissolve, reduce, all dead.When total amount reaches 45ml, put 50ml and be centrifuged Guan Zhong, 10 minutes, abandons supernatant, adds 3ml freezing media (5% DMSO (dimethyl by centrifugal 1500 turns Sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, Put-20 DEG C of 2h, then put-70 DEG C of 2h, In liquid nitrogen container).

(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: take growth preferably transfection Cell, makes cell suspension, through counting, takes 1.4 × 10 respectively⁴Cell is inoculated in 30 low sugar DMEM culture medium Han 15FBS Culture bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation, until cell quantity is decreased obviously, is cultivated 3 days After gave no count every 2 days cell change liquid, use same method to observe transfectional cell and train at hepato ZYME-SFM serum-free Support the growing state in base.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale Make after curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof "；(iii) Check chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", then Illustrate that this cell line does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA group in flow cytometry analysis cell line Body, if it has not, there is not tumor feature in explanation cell line).Chromosome karyotype analysis method is: pre-by adding in 5mL culture fluid The 250ug/ml Colchicine 100ul of heat, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, removes supernatant, hypotonic, solid Calmly, film-making, G show band post analysis karyotype；(iv) Flow cytometry: detection the 19th continuous cell line synthesizes, Division cell proportion, if its multiplication capacity substantially ratio do not build be normal cell strengthen, explanation be SV40 large T antigen integrate, The result expressed.(viii) determined dna sequence: sequenator detection routinely, shows SV40 large T antigen DNA sequence.(v) SV40 big T gene test in transfectional cell DNA: as with Immunohistochemical detection, in the nucleus of SV40 transfection, dyeing can See a large amount of brown particle, show that SV40T antigen has been integrated into intracellular；Also can be with RT-PCR method detection T antigen in cell Express, the wherein primer of T antigen: forward primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', under Trip primer (S4496) 5 '-GAA ATG CCA TCT AGT GAT-3 '；The a length of 268bp of amplified production, amplification condition is 94 DEG C, 5min, it may be assumed that (94 DEG C, 1min；55 DEG C, 1min ,-0.5 DEG C/circulation；72 DEG C, 1min) × 30, (94 DEG C, 30S；40 DEG C, 30S, 72 DEG C, 30S) × 15, amplification system is 50 μ l:[Mg²⁺]2mmol/L、dNTPs 200 μm ol/L, primer concentration 0.4 μm ol/L, Taq1U, template 5 μ l；Experimental group is with the cDNA of the 19th generation cell as template (carrying out the synthesis of cDNA the first chain, product-20 DEG C preservation with reference to commercially available cDNA the first chain synthetic agent box)；Negative control sets two Individual, do template with the cDNA of sterilized water, primary cell respectively, positive control is (reference SDS-albumen with SV40 DNA as template Enzyme K method extracts SV40 DNA, because SV40 virus is without peplos, does not use SDS rupture of membranes, takes 5 μ l and carry out 1.5% agarose Detected through gel electrophoresis, remaining-20 DEG C save backup)；(vi) mrna expression product measures: T antigen mRNA RT-PCR product is surveyed Sequence: take the amplified production of 100 μ l systems, reclaims test kit (Takara, Japan) with gel and reclaims product, take 2 μ l DNA Solution dilutes 100 times, surveys concentration, and remaining DNA and each 10 μ l of upstream and downstream primer checks order.

(VIII) SV40LT gene mediated CD4+T cell bank: screen and continue to pass on, amplification culture meets after above-mentioned qualification Immortalized cells characteristic the cell same or like with primary cell, take the difference that growth conditions is good, be in exponential phase Cell from generation to generation, is performing centrifugal separation on (1200r/min, 6min), resuspended with the frozen stock solution 0.5～1ml containing dimethyl sulfoxide Cell, cell density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, mistake At night, entering-196 DEG C of liquid nitrogen cryopreservations, the CD4+T cell bank building biological characteristics stable in this way is standby.

(4) with the preparation of CD4+T cell identity function granule: can be by CD4 molecule, the CD4 molecule of gene recombinaton and similar merit The molecule of energy is fixed on carrier by conventional chemical coupling, crosslinking, affine absorption etc. makes the granule being coated with CD4 molecule, Or directly take the replacement CD4+ cell application of intimate granule.The CD4+T cell of the present invention represents other CD4+ cells, bag Include and prepare immortalization CD4+T cell with additive method.

Two, the preparation of HIV immunologic purging device

1, the filling of cleanser

Hybridoma macrophage prepared by the present invention and CD4+T cell, after cleaning with physiological saline solution respectively, then with 1000r/min is centrifuged 5min (low speed is centrifuged in short-term), by the ratio of hybridoma macrophage strain and CD4+T cell strain be 1: 0.5～ The ratio of 3 takes sedimentation cell, loads in the 200ml hydrostatic column that acrylate etc macromolecular material is made, is fills up to 4/5, Then add cells frozen storing liquid (containing 30% hyclone, the RPMI-1640 of 12% dimethyl sulfoxide), make cell concentration reach 80%, gently Jog is even, sealing, through 4 DEG C, and 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen cryopreservations standby.Thaw During use, need cryopreservation tube to be put in 37 DEG C water-baths having been warmed up rapidly, and constantly shake, make the liquid in pipe melt rapidly Change, use after then cleaning with physiological saline solution.Wherein hybridoma macrophage and CD4+T cell are all fixed on depurator In, play the effect of absorption HIV.

2, the specification of depurator

The cell column of above-mentioned preparation is depurator, and the cylinder little for footpath, the end, footpath, top is big, or square, infundibulate, volume is 200～300ml, to import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh；Footpath sieve number at the bottom of exit Be 2.0～5.0 mesh (2.5～5.0 mesh are equivalent to 0.1～0.2 micron or 100～200 nanometers), specifically can be made into 2.0 mesh, 7 kinds of different sizes of 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh, in order to stop the HIV of 120 nanometers Viral or bigger antibacterial；Liquid outlet arranges the cell strainer that mesh number is 100 mesh (being equivalent to 4 microns), in order to stop The cell that may leach；Relief area, the beneficially stability of system circulation it is provided with between liquid entrance and mesh screen.

3, the material of depurator

Blood purification selects the macromolecular material of acrylate etc, it is desirable to good biocompatibility, hardly activating complement, no Cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Covalency can be passed through, connect The method such as branch, polymerization improves the structure of materials, the regulation inhomogeneities on surface, hydrophilic, minimizing to blood coagulation and oxidative stress Affect thus improve biocompatibility, the generation of minimizing complication.Hydrophilic gel is added, by 2 methyl-prop at depurator inner surface Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate membrane, by controlling wet-spinning procedure, can generate CA/PMB30, CA/PMB80 and CA/PMB30-80, have higher blood and cell compatibility.Some has anticoagulant make Material be solidificated on the material of carrier or depurator inner surface, can suppress blood coagulation, improve biocompatibility, also can drop Low heparin consumption, and likely realize no-rod tractor.As being aggregated in by heparin on polyacrylonitrile-polymine film, effect may More preferably, and can reduce the anaphylaxis during purification, the polyacrylonitrile surface of solidifying shell polysaccharide and heparin covalent thing also show Good blood compatibility, and the activity of pseudomonas aeruginosa can be suppressed, reduce cell-cytotoxic reaction.On cellulose acetate film altogether Valency solidification linoleic acid film, maybe will be covalently bound to polyacrylic linoleic acid and be grafted onto polysulfone membrane surface, can have more preferable group Knit the compatibility and anticoagulant effect.

Three, the preparation of plasma separator

1, preparation principle: prepare according to the molecular size of hemocyte and blood plasma components.Such as visible component (hemocyte) in blood of human body Size be: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leukocyte is divided into 5 kinds, neutral Granulocyte about 12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, small lymphocyte 6-8 μm, approximates with erythrocyte, and mononuclear cell is maximum, about 15-20 μm.Platelet is disc, diameter 1～4 microns to 7～ 8 microns, the platelet mean diameter of people is 2-4 micron, thick 0.5～1.5 micron.

2 materials: can be selected for poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc., it is desirable to good biocompatibility, activate benefit hardly Body, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can be by altogether Valency, be grafted, the method such as polymerization improves the structure of material, the regulation microinhomogeneities on surface, hydrophilic, minimizing to blood coagulation and The impact of oxidative stress thus improve sieving adequacy and biocompatibility, the generation of minimizing complication.

3, type and spec: for the profile of separator, can be prepared as cylindricality with the material such as acetate fiber or absorbent cotton as filter element Structure, it is prepared as the shapes such as flat structure as filter element with materials such as poly-vinegar non-woven fabrics；By hemocyte to be separated and blood plasma components Molecular size determines aperture.Plasma separator stable in properties involved in the present invention, good biocompatibility, height that permeability is high Molecularly Imprinted Polymer makes hollow fibre type filter, hollow-fiber film a diameter of 270～370 μm, and film thickness is 50 μm, and aperture is 0.2～0.6 μm, fibre length is 13.5～26 μm.This hole is only permitted blood plasma and is filtered, but can stop all of cell component.

Four, the component of HIV immunologic purging device

1, key member: (1) plasma separator: be used for separating mononuclear blood cell and blood plasma；(2) plasma purification device: be used for inhaling HIV in attached blood plasma.

2, additional member: include blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, electricity The part compositions such as conductance monitoring system, ultrafiltration monitoring and leakage blood monitoring.(1) blood pump (Blood Pump): be used for promoting blood to follow Ring is to maintain being smoothed out of blood purification treatment, and usual blood pump part often has rotary test speed function, to monitor the blood of patient Stream situation, therefore blood pump runner and flute pitch set and want accurately and it needs to often adjust, according to the situation of bloody path pump line, one As spacing is set as 3.2～3.3mm, can not be the most loose, blood flow detection otherwise can be caused to be forbidden；Also can not be too tight, otherwise can make Become pipe breakage.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump applied clinically, in order to hold Continue injecting heparin in sieving pipeline (patient blood), owing to the blood of patient circulates and air contact in vitro, be susceptible to Blood coagulation phenomenon, uses heparin pump to be possible to prevent the generation of blood coagulation.(3) sound pulse pressure monitoring: arterial blood pressure monitoring is mainly in order to dynamically The stopping state of monitoring blood separator micropore, additionally in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.Work as blood flow Time not enough, arterial pressure will reduce；When having blood coagulation, thrombosis, particularly during separator blockage of the micro orifice, arterial pressure will rise High；Venous pressure monitoring is used for monitoring the pressure of pipeline blood backflow, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow When deficiency and venous return syringe needle come off, venous pressure will decline, if the distortion blocking of bloody path return duct or backflow syringe needle are sent out During raw blocking, venous pressure will raise.(4) air monitering (Air Detector): be used for monitoring the air bubble of blood pathway, The principle of general ultrasonic listening, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble, inspection Examining system can drive artery and vein bloody path folder to carry out blocking blood flow, prevents the generation of danger.

In a word, on the basis of key member of the present invention and additional member, Import computer regulation and control and make operation hommization, The personalization for the treatment of, the safety of design, and modularity, automatically monitoring and regulation and control, liquid crystal display, judge that alarm is former voluntarily The blood purifying therapeutical instrument that the micro computers such as cause and ring off signal process.

Five, the connecting path of HIV immunologic purging device and using method

1, install: such as Fig. 1, with sterile working's connecting components, including plasma separator, depurator and each circulation line.

2, aerofluxus: with physiological saline solution topping up separator, depurator and each circulation line, get rid of separator, depurator and Gas in circulation line, bubble, go through, and confirms without using after gas, bubble.

3, logical liquid: arterial blood line pipe (1) is connected the arteries of HIV sufferers, the most again goes through aerofluxus The most complete, liquid stream is the most unobstructed, and avoids managing the pollution of interior flow liquid.

4, anticoagulant: inject anticoagulant (heparin) from heparin pump (2) to liquid stream, be 2500 ∪ or 20～30 ∪/kg for the first time.

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (5) connected vein blood vessel, Then opening blood pump (2), blood flow is 100～150ml/min, and such as Fig. 1, arterial blood enters through arterial blood line pipe (1) Plasma separator (4), the blood plasma separated arrives depurator (8) through circulation line (7) under the effect of blood plasma pump (6), Blood plasma to be full of, about 10 minutes, begin paying out blood plasma, through circulation line (10) flow out, synchronize to depurator (9) irrigate Blood plasma, when the blood plasma in depurator (8) has nearly flowed, starts again at perfusion blood plasma, and now depurator (9) begins paying out Blood plasma, two depurators in parallel (8), depurators 9) alternately, the blood plasma after purification is through circulation line (10) and blood plasma The hemocyte that separator (4) is separated feeds back through venous line (5) after converging.Such as Fig. 2, when blood to be separated enters blood When starching inner chamber (2) of separator (1), through the effect of valve (8), the little molecule blood plasma components (5) of micropore (3) can be passed through Enter the exocoel (6) of separator, then flow out through plasma outlet port (7), and hemocyte (4) warp of micropore (3) can not be passed through Valve (8) flows out.Such as Fig. 3, when the blood plasma containing HIV (1) enters depurator, HIV therein (1) is consolidated respectively The macrophage strain (2), the CD4+T cell (4) that are scheduled in depurator (6) are combined into macrophage phagocytic thing (3), CD4+T Cell conjugates (5), combined after HIV no longer move down, so remove blood plasma HIV, until the blood plasma being previously set Circulating load (usually 9L), treatment just ends, and whole therapeutic process is by computer control, and can detect work shape at any time State, easy to use, automatization and safety.

Six, the checking of HIV immunologic purging device effect

1, CD4+T cell strain removes the checking of HIV effect

In order to verify effect of CD4+T cell strain adsorption removal HIV, the present invention devises easy method of testing: take sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draws by centrifugation CD4+T cell that (1000r/min, 5min) precipitate respectively to 200mm Scale, then draws and is incubated after 100 DEG C dissolve at 39～42 DEG C of 0.9% standby agarose C1-4B, reach about 10mm length Scale, after putting blood sedimentation stand cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but will not stop little molecule The material of water and chemical analysis etc passes through.The 5 of the HIV sufferers that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse preserve Example blood plasma, the most about 10mL, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube upper end blank pipe, under blood sedimentation tube to be flowed through in batches Layer CD4+T cellular layer and in blood sedimentation tube flow out after, collect effluent, referred to as AIDS filter after blood plasma.Take blood before AIDS filter Blood plasma after slurry and filter, according to HIV-1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) Operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is less than 5pg/ml, measurement range 0～400pg/ml, linear model as comparison, lowest detectable limit Enclosing 0.5pg/ml～80pg/ml, in 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is the highest In 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, as absorbance ＞ 0.12 Being considered as positive, testing result (table 1) illustrates, after AIDS blood plasma filters the simple purifier containing CD4+T cell, and portion Dividing HIV to be adsorbed by CD4+T cell, after the 1st time filters, HIV total body clearance is 22.84%, after the 2nd time filters, Total body clearance is 35.31%, and after the 3rd time filters, total body clearance is 41.9%.Illustrate along with the increase filtering number of times, HIV Can constantly be removed, thus reach to treat AIDS purpose.

Table 1 AIDS blood plasma filters p24 testing result (pg/ml) before and after the simple purifier containing CD4+T cell

2, the checking of HIV effect is removed in macrophage strain

In order to verify that effect of HIV is removed in macrophage strain, the present invention devises easy method of testing: take sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draws by centrifugation the macrophage hybridoma cell strain that (1000r/min, 5min) precipitates respectively To 200mm scale, then draw and be incubated after 100 DEG C dissolve at 39～42 DEG C of 0.9% standby agarose C1-4B, reach about The long scale of 10mm, after putting blood sedimentation tube cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but will not stop little The material of the water of molecule and chemical analysis etc passes through.The acquired immune deficiency syndrome (AIDS) that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse preserve (AIDS) 5 example blood plasma, the most about 10mL of patient, before respectively taking 9mLAIDS filter, to inject blood sedimentation tube in batches (the cleanest for blood plasma Gasifying device) upper end blank pipe, wait flowing through the hybridoma macrophage strain layer of blood sedimentation tube lower floor and after flowing out in blood sedimentation tube, collect stream Go out liquid, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) pairing detection, by specification operate, detection range be 0～ 800pg/ml, sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, positive The strongest, negative reaction is colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: On ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, after returning to zero with blank control wells, survey each hole OD Value, if 2.1 times of the negative control OD value more than regulation, it is the positive.Result such as table 1, MIF detection knot in blood plasma before filter Fruit is feminine gender (or because content preserves cause degraded etc. for a long time less than detection sensitivity, blood plasma), and testing result in blood plasma after filtering It is the positive.Illustrate that macrophage hybridoma cell strain creates MIF cytokine in this process.MIF be collection cytokine, Somatomedin, hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction is sent out Wave the effect of central, various infection and active chronic inflammation disease play panimmunity function.Filter making AIDS blood plasma Before and after simple depurator while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 Antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operate, with concentration known 0pg/ml, 0.5 The p24 antigen of pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as right It is less than 5pg/ml according to, lowest detectable limit, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, 15 In min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is the highest In 0.100,1000pg/ml absorbance is not less than 1.000, is considered as positive as absorbance ＞ 0.12, result (table 2) After illustrating that AIDS blood plasma filters simple purifier, part HIV is by the phagocytosis absorption of macrophage hybridoma cell strain, after filtration Blood plasma HIV significantly reduce, through the 1st time filter after, HIV clearance rate is 20.55%, through the 2nd time filter after, HIV remove Rate is 42.83%, and p ＜ 0.01 has a significant effect, and illustrates that the increase HIV along with filtering number of times can constantly be removed, Thus reach to treat AIDS purpose.

Before and after table 1 AIDS blood plasma filters the simple purifier containing hybridoma macrophage, MIF testing result is (quantitative: pg/ml)

Table 2 AIDS blood plasma filters p24 testing result (p24:pg/ml) before and after the simple purifier containing hybridoma macrophage

In a word, above-mentioned simple experiment shows, HIV free in blood plasma can by the present invention blood purification agent (macrophage strain and CD4+T cell strain) adsorption removal, show the HIV immunologic purging utensil of the present invention to have significantly and remove controlling of blood plasma inhibition of HIV Treat effect.

Claims

1. the HIV immunologic purging device for medical domain, it is characterised in that bite huge for made hybridoma Cell strain and CD4+T cell strain are formulated in cells frozen storing liquid, the outlet that perfusion high-biocompatibility material is made Place arranges the depurator of screen cloth, makes daf molecule account for 4/5, constitutes with the separator of energy separated plasma and hemocyte The main part of purging in vitro device, for removing the HIV in blood plasma.

HIV immunologic purging device the most according to claim 1, it is characterised in that described depurator is by it The screen cloth in exit, hybridoma macrophage strain, CD4+T cell strain collectively form molecular sieve mechanical removal and thin The barrier of born of the same parents immune clearance HIV.

3. according to the arbitrary described HIV immunologic purging device of claim 1,2, it is characterised in that described only The volume changing device is 200～300ml, imports and exports and is equipped with cell screen cloth, and footpath, import department top sieve number is 800 Mesh, footpath sieve number at the bottom of exit is 2.0～5.0 mesh, and liquid outlet arranges the cell that mesh number is 100 mesh Filter screen, arranges the relief area of accelerating system stable circulation between liquid entrance and screen cloth.

HIV immunologic purging device the most according to claim 3, it is characterised in that footpath at the bottom of described exit Sieve number makes 7 kinds of 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh Different size.

5. according to the arbitrary described HIV immunologic purging device of claim 1 and 2, it is characterised in that described Prepared by the hybridoma technology that macrophage strain is merged with cell.

6. according to the arbitrary described HIV immunologic purging device of claim 1 and 2, it is characterised in that described CD4+T Cell strain is using SV40 and/or hTERT as rotaring redyeing gene, dual anti-with CD3 monoclonal antibody and/or CD28 Body is prepared as cell growth stimulant.

HIV immunologic purging device the most according to claim 1, it is characterised in that described separator is empty Core fiber film a diameter of 270～370 μm, film thickness is 50 μm, and aperture is 0.2～0.6 μm, and fiber is long Degree is 13.5～26 μm, can stop that all of cell filters.

8. the preparation for the HIV immunologic purging device cleanser of medical domain, it is characterised in that by miscellaneous Handing over tumor macrophage strain and CD4+T cell strain, clean with physiological saline solution respectively, 1000r/min is centrifuged, It is centrifuged 5min with 1000r/min again after Xi Jinging, by the ratio of hybridoma macrophage strain and CD4+T cell strain is The ratio of 1: 0.5～3 takes cell precipitation and is formulated in cells frozen storing liquid, and perfusion high-biocompatibility material is made In 200ml hydrostatic column, make depurator, make daf molecule account for 4/5, seal, through 4 DEG C, 0.5h； -20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen cryopreservations standby, thaw use time, need to rapidly by Cryopreservation tube is put in the 37 DEG C of water-baths having been warmed up, and constantly shakes, and makes the liquid in pipe melt rapidly, Then use after cleaning with physiological saline solution.

9. according to the arbitrary described HIV immunologic purging device of claim 1-8 in preparing purging in vitro device Application, it is characterised in that described purging in vitro device includes that one end of arterial blood line pipe (1) is through heparin pump (2) Being connected with plasma separator (4) with blood pump (3), plasma separator (4) is through blood plasma pump (6) and follows Endless tube road (7) depurator (8) in parallel with two, depurator (9) are connected, the most successively through circulation pipe Road (10), venous line (5) are confluxed.

Application the most according to claim 9, it is characterised in that when start shown in Fig. 1 external only During gasifying device, arterial blood enters plasma separator (4), the blood plasma separated through arterial blood line pipe (1) Depurator (8), blood plasma to be full of, about 10 is arrived through circulation line (7) under the effect of blood plasma pump (6) Minute, begin paying out blood plasma, flow out through circulation line (10), synchronize to irrigate blood plasma to depurator (9), When blood plasma in depurator (8) has nearly flowed, starting again at perfusion blood plasma, now depurator (9) leaves Begin to release blood plasma, two depurators in parallel (8), depurators 9) alternately, the blood plasma after purification is through following The hemocyte that endless tube road (10) is separated with plasma separator (4) feeds back through venous line (5) after converging.