CN106267425A - AIDS immunoadsorption therapy instrument - Google Patents

Abstract

nullA kind of AIDS immunoadsorption therapy instrument for medical domain，It is characterized in that preparation can separated plasma、Mononuclear blood cell and the blood plasma of multinucleated giant cell formed because living away from home HIV and blood separator，Preparing with hybridoma technology and exogenous gene rotaring dyeing technology respectively can be in conjunction with the hybridoma macrophage strain of HIV and CD4+T cell strain，It is formulated in cells frozen storing liquid as adsorbent，The adsorber that perfusion macromolecular material is made，With the critical component that separator collectively forms extracorporeal circulation apparatus，When blood flows through blood separator，Multinucleated giant cell containing HIV is filtered out，And then the blood plasma separated by plasma separator is when flowing through adsorber，HIV therein is adsorbed to be removed，The mononuclear blood cell that blood plasma after purification is separated with plasma separator feeds back after converging，Thus reach to remove in hemocyte、The purpose of outer HIV.

Description

AIDS immunoadsorption therapy instrument

Technical field

The present invention relates to preparation and the application of AIDS immunoadsorption therapy instrument in biomedical sector, be mainly used in acquired immune deficiency syndrome (AIDS) The removing of the inside and outside HIV (human immunodeficiency virus) of patient's hemocyte, thus reach to prevent, control and treat the purpose of acquired immune deficiency syndrome (AIDS).

Background technology

HIV (human immunodeficiency virus) is a kind of slow virus (Lentivirus) infecting human immune cells, belongs to reverse transcription sick The one of poison, diameter about 120 nanometer, the most spherical in shape.Outer virionic membrane is lipoid peplos (from host cell), and is embedded with virus Albumen gp120 and gp41.Gp41 is transmembrane protein, and gp120 is positioned at surface, and is combined by noncovalent interaction with gp41.To It is inside the sphere matrix (Matrix) formed by albumen p17, and the half-cone capsid (Capsid) that albumen p24 is formed, capsid In high electron density under Electronic Speculum, include virulent rna gene group, enzyme (reverse transcriptase, intergrase, protease) and other Composition from host cell.

After HIV enters human body, first by macrophage phagocytic, but HIV quickly changes macrophage some position interior Sour environment, creates the condition of its existence applicable, is not the most killed and the most within it breeds.Because CD4 is being subject to of HIV Body, thus in macrophage breeding HIV by its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes self hydrophobic interaction mediate retroviral cyst membrane and cell membrane fusion) (cell, monokaryon are huge to be bitten carefully to enter CD4+ cell Born of the same parents, dendritic cell etc.), in intracellular rapid propagation, produce 10 every day⁹～10¹⁰Virion, and it is normal constantly to enter other And regeneration the intracellular duplication of CD4+, manufacture more virus infected cell, make peripheral blood CD4+T cell sustaining breakdown, subtract Few.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, indirectly causes a large amount of broken of CD4+ cell by the crosslinking of cell surface CD4 molecule Bad, 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, and NK cell, huge bites Cytoactive weakens, and the synthesis of the cytokine such as IL2, IFN-γ reduces.CD4+T cell is most important immunocyte, infects Person once loses a large amount of CD4+T cell, and whole immune system will suffer deathblow, and the infection to various diseases is all lost Go resistance.HIV can also show as hiding for a long time and not showing clinical symptoms after entering host's CD4+ cell, its genome 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 integrated 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 The AIDS several months is to incubation period for many years.In the incubation period of AIDS, HIV is mainly in the macrophage and dendritic cell of lymph node Breeding, these cells are internal HIV depots, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, have silk to divide Splitting former, antigen, TNF, IL-2 and lymph element (LT) can excite HIV provirus gene multiple at the CD4+T Intracellular transcription infected System.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 cells continue course of infection.

Body can be stimulated after HIV to produce envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody.? Measuring low-level antiviral neutralizing antibody in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers are the highest, and the most protected effect of this antibody is described.But antibody can not be with the virus that retains in mononuclear phagocyte Contacting, and HIV envelope protein easily occurs antigenic variation, original antibody is ineffective, makes neutralizing antibody not play due Effect.In the latent infection stage, HIV provirus is integrated in host cell gene group, and therefore HIV will not be known by immune system Not, so only relying on autoimmune function and cannot being removed.The critically important reason of another one should be to kill according to antibody Go out, remove antigen mechanism speculate, after immune antibody is combined with antigen, if immunological effect to be produced, or pass through activation Complement, mediation ADCC effect dissolves cellular antigen, but HIV is not cellular antigen；Phagocytosis is attracted by chemotaxis Antigen is removed in cell phagocytosis, but HIV is protected in phagocyte on the contrary, breeds；Antibody has been combined neutralization and has made with antigen With, it is allowed to lose appeal, but HIV antigenic structure is changeable, often make 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 suppresses Agent: be only capable of preventing the permissive cell of not yet infected by HIV from infecting, the cell infected do not had therapeutical effect, and toxic and side effects is relatively Many, including gland plastochondria toxicity, bone marrow depression, globular anemia, granulocyte and thrombocytopenia, pancreatitis, intersect resistance in addition The generation of the property of medicine, this kind of medicine is not used alone, and mainly does drug combination, and is easily generated drug resistance mutant, causes under clinical efficacy Fall or inefficacy.(2) hiv protease inhibitor: be easily generated the toxic and side effects such as drug induced hepatic injury, lipid metabolic disorder and drug resistance Property.(3) hiv integrase inhibitor: integrated with host cell DNA by suppression inhibition of HIV DNA and play a role, reverse with HIV Transcriptase inhibitors and protease inhibitor associating use simultaneously and antiviral are had synergism.(4) suppression inhibition of HIV enters suppression Agent: include block gp120 with CD4 be combined, block HIV be combined with accessory receptor, act on gp41 film subunit and act on T pouring Bar cell surface CC-chemokine receptor 5 (CCR5) blocks HIV and enters host cell, but liver and heart are had side effect.(5) Cytokine therapy: be mainly used in regulatory T-cell dynamic equilibrium.(6) HIV vaccine treatment: due to the particularity of HIV, as intrinsic Immunity is not enough to resist HIV and targeting destroys immune system, and virus mutation is rapid, causes the most not yet developing real safety Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, does including antisense technology, RNA bait, RNA Disturb, intrabody, dominant negative mutant, suicide gene etc., but enter the II clinical trial phase stage gene therapy almost No.(8) monoclonal antibody passive immunization therapy: reduce the susceptibility of HIV by lowering CD4+T cell surface CCR5, prolong The progress of slow acquired immune deficiency syndrome (AIDS) and the diffusion of minimizing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10 are applied to HIV person to be had Good toleration and safety, can delay but can not stop virus bounce-back (9) adoptive immunity cell therapy: external in a large number Virus a large amount of amplification can be caused when cultivating CD4+T cell autologous for HIV, increase the CD4+T cell quantity that virus infects, and feed back CD4+T cell may increase the place of internal virus replication, causes virus load to rebound, and on the whole, adoptive immunity is thin Born of the same parents treat without obvious toxic and side effects, also do not obtain satisfied therapeutic effect.

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, averagely Life-span is generally about 7 days, but some T cell particularly after immortality chemical conversion cell line (strain) can long-term surviving, infinitely expand. Foreign literature 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 SV40T 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 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 membrane Type is with the inhibitory action mechanism of research heparin for vascular smooth muscle.Su etc. utilize the superficial cell strain converted through SV40, structure Build cell model to analyze the regulating and controlling effect of epithelial cell internal protein synthesis.The cuticulated epithelium that Miquel etc. convert with SV40 is thin Born of the same parents' strain, as the cell adhesion of cell model research laminin，LN 5 mediation.The prostatitis converted through SV40 such as Webber Glandular epithelium strain studies physiological function and the secretory function of prostate epithelial cell as cell model.Racusen etc. use Convert renal cells model through Ad12-SV40 and study damage and the disease of proximal convoluted tubule.The SV40 such as Hougton turns Change set up Bone marrow Stromal cell as cell model with research under certain condition of culture, cell have to adipose cell with become The potential of the two-way differentiation of osteocyte, studies osteoporotic mechanism further.Foreign study is it is also shown that import exogenous human end Human telomerase reverse transcriptase (hTERT) can make cell keep normal phenotype and differentiating characteristic.HTERT has the most been utilized to be successfully established The immortalized cell line of some cell, keeps that chromosome stabilityX, differentiation be normal, contact inhibition, relative without oncogenicity etc. substantially Normal growth characteristics.In stomatology field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes immortality Changing people's Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number reaches more than 150 times, Cell all shows original biological characteristics, can express the associated protein of derived cell after inducing culture.Kitagawa etc. Transfection hTERT establishes people cementoblast system, and cell multiplication reaches more than 200 times, cell differentiation mark such as alkaline phosphatase Enzyme, type i collagen etc. are expressed stable.Because of the needs of research work, almost every kind of disease has respective cell model.Such as diabetes Cell model, cancer cell model, transgenic cell model, climacteric syndrome cell model, endometrial cell model, Epilepsy cell model, E-Cell models, alcoholic dementia cell model, cerebral edema cell model etc..So, CD4+ can be prepared T cell strain, after mass propgation, for preparing the adsorber for the treatment of acquired immune deficiency syndrome (AIDS), with the absorption of CD4+T cell, removes in blood plasma HIV, simultaneously by being defeated by reactor the cytokine of the CD4+T cell produced, treats HIV sufferers.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophilic granulocyte in peripheral blood, macrophagocyte Being the mononuclear cell in blood and the macrophage in multiple organ, tissue, both constitute mononuclear phagocyte system.Mononuclear cell Formed by the monocyte precursor Development And Differentiation in bone marrow, account for the 3%-8% of blood middle leukocytes sum, its volume relatively lymph Cell is bigger, and mononuclear cell the most only stops 12-24 hour, subsequently into connective tissue or organ, reaches maturity as huge Phagocyte, macrophage is the differentiation of mononuclear phagocyte system camber, ripe cell type, has stronger phagocytosis merit Can, wandering macrophage is more than mononuclear cell several times, lasts a long time, some months of can surviving in the tissue, the macrophage settled down Having different titles, be Kupffer Cell, be microglia in brain, be osteoclast etc. in bone in liver, it expresses Fc Receptor, C3b receptor and CDl4, play defense function in inherent immunity, is also that the professional antigen participating in adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of HIV (human immunodeficiency virus) (HIV), after HIV enters human body, first suffers huge biting carefully The phagocytosis of born of the same parents, but HIV quickly changes the sour environment at macrophage some position interior, creates condition of its existence applicable, The most 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 adsorber 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 AIDS immunoadsorption therapy instrument；Another object is intended to provide AIDS immunoadsorption to control Treat preparation and the application process of instrument.

The object of the present invention is achieved like this: it is thin that preparation can filter the large volume containing HIV that many cells are combined into The blood separator of born of the same parents and mononuclear blood cell and the plasma separator of blood plasma can be separated, build with the method for exogenous gene transfection CD4+T cell strain and/or using the peculiar molecular antibody of CD4+T cell surface as cell growth stimulant amplification, with hybridoma skill Art preparation had not only retained former macrophage feature but also can the hybridoma macrophage strain of indeterminate growth expand parallel, took CD4+T cell Strain and macrophage strain are formulated in cells frozen storing liquid, container that perfusion high-biocompatibility material is made and prepare and can prevent cell And fragment leaches and can swallow for cell strain, adsorb HIV provides the HIV adsorber in place, wherein CD4+T cell strain and huge bite Cell strain is all fixed in adsorber, plays the effect of absorption HIV, prepared HIV adsorber and then separate with blood, blood plasma Device combination and additional computer regulatory process and prepare AIDS immunoadsorption therapy instrument, the blood in extracorporeal circulation is treated in instrument Blood separator filter many nuclear blood cells of large volume, and then be divided into blood plasma and mononuclear blood cell, blood plasma by plasma separator Feed back after converging with mononuclear blood cell after adsorber filters HIV.

The technological core of the present invention is made up of blood, plasma separator and HIV adsorber, wherein the aperture of blood separator Model is 150～250 μm, 50～150 μm, 15～40 μm, 8～15 μm, 5～8 μm, 3～5 μm, 1～2 μm, can filter in blood The plastidogenetic multinucleated giant cell of HIV or many cells polymer；The single blood that plasma separator can separate medium volume is thin Born of the same parents and blood plasma；Adsorbent in HIV adsorber is made up of CD4+T cell strain and macrophage strain, is formulated in cells frozen storing liquid Can save backup with prolonged cold, because the CD4 molecule of CD4+T cell surface is the receptor of HIV and become the permissive cell of HIV, HIV, adsorbed HIV can be adsorbed when meeting with HIV be fixed in adsorber with CD4+T cell, bite because hybridoma is huge again thin The natural phagocytosis characteristic of born of the same parents, consequently also can be fixed in adsorber by phagocytosis when HIV meets therewith, so at acquired immune deficiency syndrome (AIDS) blood In the extracorporeal circulation of liquid purification treatment, first the large volume cell containing a large amount of HIV is filtered by blood separator, and blood plasma middle reaches From HIV be adsorbed device adsorption removal, the mononuclear blood cell of the blood plasma after purification and medium volume feeds back after converging, thus removes It is combined in cell surface and/or intracellular HIV and is free on the HIV of blood plasma.The present invention replaces with the method for the external HIV of filtering In generation, cannot effectively kill the routine internal anti-reverse transcription drug treatment of HIV for a long time.

Detailed description of the invention

Fig. 1 is the application schematic diagram of the AIDS immunoadsorption therapy instrument proposed according to the present invention.

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

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

Fig. 4 is the internal structure schematic diagram of the adsorber proposed according to the present invention.

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, the other end through heparin and blood pump (2) with contain The blood separator (3) having waste liquid outlet (5) is connected, and blood separator (3) exports (4), blood pump (6), circulation pipe through blood Road (7) is connected with plasma separator (8), the plasma outlet port of plasma separator (8) through blood plasma pump (9) and blood vessel (10) with two Adsorber (11) in parallel is connected with adsorber (12), the export pipeline (13) of two adsorbers and the blood of plasma separator (8) Cell outlet pipeline (14) circulates through venous line (15) fluid confluence after converging.

Fig. 2 represents the internal structure of the blood separator (3) in Fig. 1.In Fig. 2, the tube wall of the inner chamber (2) of separator (1) On have a lot of micropore (3), multinucleated giant cell (4) micropore (3) can not be filtered and be delayed at inner chamber (2), thus can be eliminated, energy By in micropore (3), the mononuclear blood cell (5) of small size enter exocoel (6), then flow out through outlet (7), and then through Fig. 1 Shown plasma separator (8) isolates hemocyte and blood plasma.

Fig. 3 represents the internal structure of the plasma separator (8) in Fig. 1.In Fig. 3,1 is plasma separator, and 2 is that blood plasma separates Device inner chamber, 3 is the micropore on plasma separator inner chamber tube wall, and 4 is the mononuclear blood cell that can not pass through micropore (3), and 5 is to pass through The blood plasma chemical analysis of micropore (3), 6 is plasma separator exocoel, and 7 is blood plasma flow export, and 8 is that to have the blood of switchable valve thin Born of the same parents export.

In Fig. 4,2,4 are respectively fixed on the macrophage strain in depurator (6) and CD4+T cell strain；1 is free HIV；3,5 respectively HIV are delayed at the conjugate in depurator (6) with macrophage strain and CD4+T cell strain after being combined, 7 is the most combined and descending HIV.

Below in conjunction with Fig. 1, Fig. 2, Fig. 3 and Fig. 4, the embodiment of the AIDS immunoadsorption therapy instrument that the present invention proposes is made Detailed description.

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 the lymphocyte separated from blood with density－gradient centrifuga－tion method and monokaryon is huge bites Cell.Concrete grammar is: the White Blood Cells Concentrate buying Blood Center or the Cord blood preserved for scientific research, takes 2mL specimen, PBS 6mL anticoagulation dropper, by hemodilution 2～3 times, is fully slowly superimposed on along tube wall after mixing that to have added 4mL lymph thin by liquid Born of the same parents separate in the 10mL centrifuge tube of liquid horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；It is divided in pipe after Li Xin 3 layers, upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, in upper, middle level Interface has the white cloud and mist layer narrow band based on mononuclearcell to be 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 supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new life Sanguis Bovis seu Bubali + 2mmol/LEDTA clearly, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add counted under microscope 4 on blood counting chamber Cell (PBMC) sum in individual block plaid.

(2) single core histiocyte: provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, its preparation method is: the 1. acquisition of spleen tissue and transhipment: in the approval of reason committee and patient's informed consent Under, take the spleen specimen tissue of excision, shred into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to rapidly cell culture chamber.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in-house blood and to ensure the aseptic of tissue.Machine Tool grinds spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 liquid.By 200 mesh stainless steel filtering net mistakes Filter is outstanding is mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing erythrocyte, lymphocyte, Macrophage 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, adds Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), removes the splitting erythrocyte fragment in supernatant, centrifugal 3 times of PBS washing, RPMI-1640 washing from The heart 1 time, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, now, mainly contains in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as cultivation Cell stock solution, Trypan Blue judges vigor and counts, and adjusting cell concentration with RPMI-1640 liquid is (3～5) × 10⁶/ L, will Adjusting the cell suspension inoculation of concentration in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2～3h, under phase contrast microscope observe form.The digestion of adherent spleen tissue macrophage: adherent spleen tissue is huge to be bitten The digestion of cell: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, and the washing of gained cell suspension is centrifugal (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or the discarded specimen of Post operation carries Take 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.Reason committee member Can ratify with under patient's informed consent, treating excess syndrome tests the residue amniotic fluid after diagnosis report, villus cell, selects exponential phase cell 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, condition of culture etc., typically Single core blood cell (PBMC) or single core histiocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method Culture dish in, in 37 DEG C, 5%CO₂Cell culture incubator (Themo electro corporation CLASS 100, beautiful State) in hatch 2h, after mononuclearcell is adherent, inhale abandon upper strata suspension cell (cell beyond macrophage be difficult to adherent and Remove with upper liquid), 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.Amniotic fluid is thin Born of the same parents, villus cell are cultivated 1～7 day, occur that cell growth clone, cell growth converge rate and reach the exponential phase of 60～80% 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: (Germany U.S. sky Ni is biological for CD4 immunomagnetic beads Technology Co., Ltd.)；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 immunity Magnetic bead sorting method: cell suspension is divided equally to two 1.5mLEppendorf pipes, centrifugal (300r/min, 20 DEG C) 10min, discards Supernatant, 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, with 500uLBuffer rinses, and by 500uL cell suspension by detached dowel, rinses detached dowel repetitive operation 3 times with 500uLBuffer, Collect effluent, containing non-cd4 cell in effluent, in separator, take out detached dowel, divide with 1000uLBuffer pressure flush From post, collecting effluent, this is that (cell viability detects cd4 cell: takes 15uL cell suspension before and after cell purification respectively and waits body Long-pending trypan blue solution mixing, the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, calculates 200 The percentage ratio of living cells in individual cell).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, sheep Sort in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, then gained Cell includes mononuclear cell and macrophage；But because the mononuclear cell life-span is short, only survives 1 day in peripheral blood and can not show a candle to huge biting Cell is prone to 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's CDl4 immunomagnetic beads test kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead is with special Opposite sex target cell one mononuclear cell combines: every 1 × 10⁸Individual PBMC adds magnetic bead and the 800uL buffering of 200uL coupling CDl4 antibody Liquid (containing 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 single cell 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 by above-mentioned cell suspension in people's cell separation post immediately, with 0.5mL wash buffer cell Detached dowel 3 times.After to be rinsed, add 1mL buffer, emigrated cells detached dowel from magnetic frame, quickly promote with pin post, Go out the cell combined with CDl4 antibody one magnetic bead in detached dowel, be 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%C0: 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 Method: CDl4 labelling: take PBMC, adjusts with buffer (bovine serum albumin 2.5mL and 2mol/LEDTA 0.5mL containing 10%) Whole cell density is 1 × 10⁸/ mL, adds CDl4+-FITC antibody 100uL, 4 DEG C of lucifuge labellings in every milliliter of cell suspension 18min, then addition 1mL streaming buffer is to terminate dyeing in centrifuge tube, PBs washs 3 times, with the PBS containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation at flow cytometer (BD FAcsAria II, U.S.) upper sorting, according to the fluorescence intensity of CDl4 antibody, the relative size of cell and the relative particle of cell and interior The complexity of portion's structure, collects 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 cattle Jinshi City Hao on daytime ocean biological product science and technology responsibility company limited purchased by serum (FBS)；DMSO (-methyl sulfoxide) is the examination of domestic analytical pure Agent.

(2) myeloma cell prepares: merges and takes out the myeloma cell (SP2/ that a pipe is frozen the last week in liquid nitrogen container 0), it is immediately placed in hot water and thaws that (applying most is Sp2/0 cell strain, and this cell strain growth and fusion efficiencies are the best, and itself is not Secreting any heavy chain immunoglobulin or light chain, the Seedling height scale of cell is 9 × 10⁵/ ml, the doubling time be 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 The NCI-H929 human myeloma cell strain that cell bank is introduced).Adding appropriate complete culture solution after thawing, 1000r/m is centrifuged 3min； It is repeated 1 times.Precipitate is moved in Tissue Culture Flask, add DMEM culture fluid, put CO2 incubator and cultivate, within 3-4 days, once pass Generation or amplification culture, adjust cell state in merging first 24 hours, it is ensured that before merging, cellular morphology is good, it is vigorous to grow.Add Appropriate basal medium is in centrifuge tube, and 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 with basal medium Adjust total cell and count to 1 × 10⁸～2 × 10⁸Merge for cell.Blue dyeing phase-contrast microscopy, viable count is expected with platform Should be higher than that 80% for qualified.

(4) cell merges: by CDl4 cell (mononuclear phagocyte) with myeloma cell with 10: the ratio of 1-5: 1 adds In centrifuge tube, 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 the 50% of 1000 μ L of preheating under aseptic condition after taking-up in 37 DEG C of water-baths PEG3000 is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, afterwards by the basis training of the 25mL of preheating Support base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, rotating centrifugal pipe lightly during adding, then stand In 37 DEG C of water-bath 10min, 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T culture medium.Suitably it is inoculated into after mixing In 96 well culture plates, it 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 Only have hybridoma after it and can grow division, now discard HAT culture medium, change complete medium.Cell clone grows When area reaches 1/10 cell hole, culture supernatant, selection is gone to have the culture hole of the hybridoma cell strain that growth conditions is good, aobvious The position of labelling cell strain growth, size under micro mirror, using aseptic rifle head to draw cell clone in the position of mark has to new In the culture hole of complete medium, doubling dilution counts hole to below the most successively, 37 DEG C, cultivates one week left side in 5%CO2 incubator The right side, basis of microscopic observation cell growth status, when cell clone covers with to hole floor space more than 1/10, take in cell or cultivation Clear detection hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain phagocytosis antibacterial Function detection: macrophage is hanged with staphylococcus or Candida albicans Liquid mixing incubation, smear, fixing, serge blue liquid dyes, and in oil Microscopic observation phagocytosis situation, counts phagocytosis antibacterial and does not gulps down Bite the macrophage number ratio of antibacterial, to swallow antibacterial function strong macrophage alternately positive clone strain.

2. hybridoma macrophage strain phagocytosis HIV Function detection: take acquired immune deficiency syndrome (AIDS) (AIDS) patient's of Disease Control and Prevention Center's preservation After blood plasma is mixed with hybridoma macrophage strain, separates cell strain, PBS 3 times, measure the phagocyte strain through cracking The function of phagocytosis 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, 40pg/ml, The p24 antigen of 80pg/ml is less than 5pg/ml, measurement range 0～400pg/ml, the range of linearity as comparison, lowest detectable limit In 0.5pg/ml～80pg/ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, is recognized as absorbance ＞ 0.12 For being the positive.Concrete test kit description of pressing operates.

3. the strain of hybridoma macrophage produces macrophage cytokines detection: with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) by specification operates, and detection range is 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, and the positive is the strongest, negative Reaction for colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: at ELISA detector On, 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 regulation 2.1 times of negative control OD value, are the positive, specifically press the operation of test kit description.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, in various infection and active chronic inflammation disease play panimmunity function.

According to testing result, the cell clone selected in the culture hole with stronger macrophage function repeats next Wheel dilution is cultivated, and repeats 2-3 wheel, takes out, proceed to culture bottle mass propgation after detection function-stable.

(6) preservation of hybridoma macrophage strain and recovery: preserve first 12 hours and adjust cell growth state, take one bottle of life Making cell suspension after long vigorous, that form is good cell, suitably 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, and-70 DEG C refrigerator overnight, puts into cryopreservation tube in liquid nitrogen container after taking-up and saves backup.The hot water of about 40 DEG C is got out before recovery, will Cryopreservation tube carefully takes out from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, is centrifuged at 1000r/min after defrosting 5min, opens cryopreservation tube under aseptic condition in superclean bench, and the cell complete culture solution after thawing washed once, then It is centrifuged 5min, supernatant discarded, in case making amplification culture at 1000r/min.

6, prepared by hybridoma macrophage strain treatment cell

The i.e. amplification cultivation of hybridoma macrophage strain.Move after using complete culture solution the most resuspended above-mentioned cell precipitation Enter in culture 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 steady Fixed.Continue in several bottles, carry out extensive industrialization to 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 (warp 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 went out The lymphocyte that HIV strain of living is immune；3. the T lymphocyte series (strain) directly bought from businessman；4. preserve for scientific research Cord blood lymphocytes cell (through inactivation HIV immunity)；The most directly take from the peripheral blood lymphocyte of HIV-1 the infected (for certainly Body), 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 Fluorescein CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company)；Lymphocyte separation medium (Shanghai perseverance letter biochemistry Reagent company limited)；Ethylenediaminetetraacetic acid (EDTA), 0.2% Trypan Blue liquid (Shanghai raw work biotechnology service public affairs Department)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany)； EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. separation (the density gradient of mononuclearcell (PBMC) Centrifuging): the aseptic 20mL venous blood that takes, and heparin sodium (500IU/mL) 2mL anticoagulant；PBS liquid is by hemodilution 2～3 times, fully After mixing, 6mL anticoagulant venous blood dropper is slowly superimposed on along tube wall and has added the 10mL of 4mL lymphocyte separation medium and be centrifuged Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in pipe；Be divided into 3 layers after Li Xin in pipe, upper strata be blood plasma and PBS liquid, lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, has one with single in upper, interface, middle level The white cloud and mist layer narrow band that nucleus is main is PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC and inserts another 50mL In centrifuge tube, add 5 times and be centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandon supernatant 50mLPBS re-suspended cell, from The heart (350r/min, 20 DEG C) 15min, abandons supernatant, add Buffer (PBS+0.5% new-born calf serum+2mmol/LEDTA, PH7.2) 2mL re-suspended cell, takes the cell that 15uL cell suspension adds on blood counting chamber in 4 block plaid of counted under microscope (PBMC) sum.3. CD4+T cell and CD8+T cell is isolated and purified: PBMC cell suspension is divided equally to two 1.5mLEppendorf manages, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, and the every 80uLBuffer of re-suspended cell contains cell Several 10⁷Individual, every 10⁷Individual cell adds 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, 4～8 DEG C of hatchings 15min, uses 1mLBuffer washed cell, and centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer is resuspended carefully Born of the same parents, are placed on MS detached dowel in the magnetic field of MACS separator, rinse with 500uLBuffer, by 500uL cell suspension by dividing From post, rinse detached dowel repetitive operation 3 times with 500uLBuffer, collect effluent, containing non-CD4+T lymphocyte in effluent Or non-CD8+T lymphocyte, in separator, take out detached dowel, with 1000uLBuffer pressure flush detached dowel, collect and flow out Liquid, this is CD4+T lymphocyte or (the cell viability detection: take 15uL cell before and after cell purification respectively and hang of CD8+T lymphocyte 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, Calculate the percentage ratio of living cells in 200 cells).

3, amplification in vitro CD4+T cell

Document is had to report the stimulant utilizing the monoclonal antibody of T cell surface C D3 molecule to grow, great Liang Pei as cell After supporting the T cell that HIV sufferers separates, feed back as self therapeutic cells.But HIV is also with the cultivation of HIV cell Breeding 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 simultaneously CD3 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, training by the method Support and within 4 weeks, be achieved with the amplification of 1000 times, and in the cell mass of amplification, CD4+/CD8+T all can expand that (CD4+T cell is more in a large number Substantially).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 train as stimulant Support HIV person's PERIPHERAL BLOOD MONONUCLEAR CELL (lymphocyte), substantial amounts of CD4+T cell, and the CD4+T expanded can be expanded Cell has 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 and carrier pLXSNneo, connects hTERT and pLXSNneo separated through PCR amplification, gel electrophoresis with Ligation Mix Digestion products, builds pLXSNneo-hTERT recon, converts 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, screening cellular morphology, growth curve, dyeing The test of body caryogram, nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemical staining, thin Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic person same or like with primary cell as hTERT immortality The CD4+T cell changed.

With the method for SV40 immortalization CD4+T cell it is: connect simultaneously through BamHI enzyme action with T4DNA 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 amp-R with amplification, purification picking Bacterium colony extracting plasmid, imports the T lymphocyte of In vitro culture with lipofection, makes the DNA integration of recon and cell, with The cell containing positive recombinant of G418 screening, passes on, amplification culture, screening cellular morphology, cell growth curve, chromosome SV40 big T gene test in the test of caryogram, nude mice tumorigenesis, transfectional cell DNA, mrna expression product measure and determined dna sequence Result meets immortalized cells characteristic person same or like with the primary cell CD4+T cell as SV40 immortalization.

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

(I) extraction of hTERT: (i) enzyme action pClneo-hTERT:hTERT be positioned at the EcoRI of plasmid pClneo-hTERT with 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, adds restriction Property restriction endonuclease EcoR I and each 0.5ul of Xho I, 37 DEG C of incubation 1h, 75 DEG C heating 15min, inactivator, add 5uL electrophoresis sample-adding Buffer (also can by add 0.5mol/L EDTA) terminates reaction, routinely after PCR method amplification hTERT, collect amplified matter with Standby electrophoresis.(ii) hTERT electrophoresis: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours into and seals Gel casting platform on, plug sample comb, after gelling is solid, from glue platform, removes envelope band, extracts comb, put into added with Enough in the electrophoresis tank of electrophoretic buffer, buffer exceeds gel surface about 1mm, prepares with 10 appropriate × sample loading buffer HTERT enzyme action sample, then adds sample in sample well with pipettor, and does suitable standard control thing simultaneously, connects electricity Pole, make hTERT face south Ghandler motion move, then under the voltage of 1-10V/cm (80V) gel electrophoresis to being sufficiently separated hTERT fragment During distance (30min), close power supply.(iii) hTERT purification and recovery: separate hTERT band from agarose: purple at long wave Under outer light source, the gel-tape containing target hTERT fragment is cut in loading bag filter, in bag filter, add 2ml running buffer Liquid, is allowed to submergence gel, and empties steam bubble, and bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), adds suitable Amount buffer, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treat that hTERT all moves under uviol lamp Going out gel, change direction of an electric field and continue energising 1 minute, from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds Entering 1.5 times of volume n-butyl alcohol, EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, So repeating secondary, add equal-volume phenol chloroform (2) and extract 2 times in the solution of lower floor hTERT, supernatant proceeds to another 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol of addition in Eppendorf pipe, in 20 DEG C overnight, 12000g, 4 DEG C Under be centrifuged 10 minutes, obtain hTERT precipitation, abandon supernatant, after adding 70% washing with alcohol 2 times, abandon dry ethanol, 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 from gel Separate, be 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 110mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C incubation 24h, builds 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 greatly Most coli strains, operating process is summarized as follows: one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16～20h (such as bacillus coli DH 5 2), or the 16～20h overnight culture that 1ml is fresh, forward to one containing 100mlLB culture medium 1L or In 500ml flask, cultivate about 2～3h (rotary shakers 200～300r/min) in 37 DEG C of violent shakings, survey every 20～30min Amount OD600 value ≈ 0.4, aseptically transfers to one, in ice-cold 50ml polypropylene centrifuge tube, at ice by antibacterial Upper placement 10～20min, is centrifuged with 4000r/min with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) in 4 DEG C 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, uses with 10ml The 0.1mMCaCl of ice pre-cooling₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding turn Head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace of final residual Culture fluid flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, at this point it is possible to rapidly Cell being distributed into aliquot, freezes in liquid nitrogen ,-70 DEG C of storages are standby.(ii) with competence escherichia coli purification, amplification PLXSNneo-hTERT recon: take 200 μ l from every kind of competent cell suspension with the sterile pipette tip of cooling and transfer to nothing In the microcentrifugal tube of bacterium, often pipe adds DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng), rotates gently with mixed Even content, places 30min in ice, centrifuge tube is put into pre-heating to the test tube rack in the circulator bath of 40 DEG C, places 90s～2min, does not shake test tube, quickly transfers in ice bath by pipe, makes cell cooling 1～2min, and every centrifuge tube adds 800 μ LSOC culture medium, is warmed to 37 DEG C with water-bath by culture medium, is then transferred to by pipe on 37 DEG C of shaking tables, and incubation 45min makes antibacterial Recovery, and antibiotic-resistance marker's gene of expression plasmid coding, turn by proper volume (every 90mm flat board is up to 200 μ l) The competent cell changed is transferred to, in the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, flat board is placed in room temperature Absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, after 12～16h, may occur in which bacterium colony.(iii) screen, expand recon: use Sterile toothpick or disinfection inoculation pin select single colony inoculation in LB culture medium aseptic for 5mL or rich medium (such as super meat Soup or TB super broth culture medium) in, after overnight incubation, it is then added to 500mL containing LB culture medium (containing suitable antibiotic) In 2L flask, cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4, for improving yield, should use surface area relatively big and band deflection plate Flask to increase venting quality as far as possible, shaking speed should be greater than 400r/min), in 4 DEG C, 6000g is centrifuged 10min, uses 4mL GTL The resuspended precipitation of solution, and transfer in the high speed centrifugation pipe of a volume >=20mL that (bacterial precipitation can be at-20 DEG C or-70 DEG C Indefinite duration preserves), add the GTE solution containing 25mg/mL lysozyme that 1mL newly joins, resuspended precipitation, place 10min in room temperature, add Enter 10mL and newly join NaOH/SDS solution, and mixing, until liquid becomes homogeneous, limpid and thickness, is placed on ice gently 10min, adds 7.5mL acetic acid solution, is gently mixed with suction pipe until viscosity declines and formed big precipitation, places on ice 10min, in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant in another clean centrifuge tube, if having visible Drift 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, in room Temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs precipitation gently, the ofest short duration the most centrifugal, sucks ethanol, very Empty dry (precipitation can be 4 DEG C of long-term preservations).(iv) qualification of recombiant plasmid and amplification: the single bacterium colony on picking plate, inoculation In 3ml is containing 100ug/ml ampicillin LB culture medium, 37 DEG C, 250r/min shaking table is cultivated, collection culture after 14h, 4 DEG C, 10000r/min be centrifuged 5min, extract in a small amount and purification of Recombinant plasmid by test kit description；Double with EcoRI and HindIII Enzyme action recombiant plasmid reaction system: each 0.5ul of restricted enzyme, 10 × buffer 2ul, recombiant plasmid 10ul, add water and supply To 20ul, 37 DEG C of enzyme action 1h.Digestion products carries out 0.8% sepharose electrophoresis, time 30min under 80V voltage conditions, and gel becomes As system is taken pictures；Measure the sequence of recombiant plasmid routinely；Recombiant plasmid, will be containing this matter after enzyme action, order-checking are identified accurately The microbionation of grain in LB culture fluid, amplification cultivation, carry out heavy dose of plasmid by heavy dose of plasmid extraction test kit description Extracting and purifying, ultraviolet spectrophotometer is standby after measuring plasmid concentration and purity.

(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, 20% hyclone In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, Typically it is 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, from The heart, removes 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, left at room temperature 45min, trains with serum-free Nutrient solution washs above-mentioned T lymphocyte 2 times.1ml serum-free is added in Lipofectamine-pLXSNneo-hTERT mixture Culture fluid, mixes gently, drops in above-mentioned T lymphocyte, and (hyclone concentration is 20ml/ to add 1ml serum-free medium L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues to cultivate 20h, discards culture fluid, and changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days select living cells make expand After cultivation, then strengthen G418 concentration to 800mg L-¹, will be able to continue by the cell of stable growth in the G418 environment of high concentration Carry out amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If cell increases Long slow, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches Transferring in 75ml culture bottle during 14ml, every 2-3 week adds 5-10ml fresh culture.Cell was cultivated to 9-10 week the (the about the 75th Generation), still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, and dead cell (passes through less than 10% The scale reading culture vessel judges the increase situation of cell quantity；Dead cell and living cells is differentiated by trypan blue staining. Because normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not enter intracellular；And loss of activity is thin Born of the same parents, the permeability of after birth increases, can be dyed blueness by trypan blue, can determine whether as cell the most dead.Method is to draw weekly one Quantitative suspension culture, mixes rearmounted room temperature 5～10 minutes, then makes cell sheet with Trypan Blue agent, aobvious Count 1000 total cellular score under micro mirror, calculate dead cell and the percentage ratio of non-staining living cells of coloring).Hereafter along with training Supporting increase and the prolongation of incubation time of algebraically, the increase of cell quantity is slack-off, dead cell gets more and more, until cell is no longer Increase, even dissolve, reduce, all death.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 points Clock, after abandoning supernatant, adds 3ml freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, then Frozen in-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).

(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 and trains containing 15FBS low sugar DMEM culture medium Support bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation is until cell quantity is decreased obviously, every after cultivating 3 days The cell giving no count every 2 days changes liquid, uses same method to observe transfectional cell at hepato ZYME-SFM serum-free medium In growing state.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and makes 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 this cell is described System does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA colony in flow cytometry analysis cell line, if do not had Having, there is not tumor feature in explanation cell line yet).Chromosome karyotype analysis method is: by adding preheating in 5mL culture fluid 250ug/ml Colchicine 100ul, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, fixing, film-making, G shows band post analysis karyotype；(iv) Flow cytometry: the cell ratio synthesizing, dividing in detection the 19th continuous cell line Example, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is the result that hTERT integrates, expresses.(vii) Determined dna sequence: sequenator detection routinely, shows hTERT gene order.In (v) transfectional cell DNA hTERT detection: as with Immunohistochemical detection, in the nucleus of hTERT transfection, the visible a large amount of brown particles of dyeing, show that hTERT has been integrated into carefully Intracellular；(vi) mrna expression product measures: takes the pcr amplification product of 100 μ l systems, reclaims test kit (Takara, day with gel This) reclaim product, take 2 μ l DNA solutions and dilute 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer surveys Sequence.

(VIII) hTERT mediates CD4+T cell bank: screen and continue to pass on, amplification culture meets forever after above-mentioned qualification OEG cell 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), with the frozen stock solution 0.5～1ml re-suspended cell containing dimethyl sulfoxide, carefully Born of the same parents' 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 and freeze Depositing, 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 electrophoresis: take Electrophoresis level agarose is made into 10% agarose gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs sample Product are combed, and remove envelope band, extract comb, put into the electrophoresis tank added with enough electrophoretic buffers after gelling is solid from glue platform In, buffer exceeds gel surface about 1mm, prepares DNA sample with 10 appropriate × sample loading buffer, then with pipettor by sample Product add in sample well, and do suitable DNA molecular amount standard control thing simultaneously, connect electrode, make the DNA Ghandler motion that faces south move, Under the voltage of 1-10V/cm gel, electrophoresis is to when being sufficiently separated the distance of DNA fragmentation, closes power supply.(iii) divide from agarose From about 2600bp SV40 large T antigen DNA: (use long wave ultraviolet light source to prevent DNA under 300-360nm long wave ultraviolet light source Damage) gel-tape containing target DNA fragments is cut in loading bag filter, in bag filter, add 2ml electrophoretic buffer, make Submergence gel, and empty steam bubble, bag filter level put into electrophoresis tank (length direction is parallel with electrophoresis), add appropriate buffering Liquid, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treats that DNA all removes gel, change under uviol lamp Energising 1 minute is continued in changed electric field direction, and from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of bodies Long-pending n-butyl alcohol, mixing extracting removes EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, so repeats two Secondary, in the solution of lower floor speech DNA, add equal-volume phenol chloroform (2) extract 2 times, supernatant proceeds 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, be centrifuged 10 minutes at 4 DEG C, DNA precipitation, abandon supernatant, abandon dry ethanol after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.Additionally, can also be used with Target DNA fragment is separated from gel, is purified by low melting-point agarose gel method, DNA filter membrane inserted sheet method etc..

(II) connection of SV40 large T antigen DNA and pcDNA3.1 genophore: take 9 μ l above-mentioned DNA composition (0.1-5 μ g), 10 μ l 2 × connection buffer, 1 μ l 10mmol/L ATP, T4 DNA ligase (20～500 sticky end unit) or large intestine bars Bacterium DNA ligase, pcDNA3.1 empty carrier mix, and 15 DEG C of incubation 24h are 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 greatly Most coli strains, operating process is summarized as follows: one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16～20h (such as bacillus coli DH 5 2), or the 16～20h overnight culture that 1ml is fresh, forward to one containing 100mlLB culture medium 1L or In 500ml flask, cultivate about 2～3h (rotary shakers 200～300r/min) in 37 DEG C of violent shakings, survey every 20～30min Amount OD600 value ≈ 0.4, aseptically transfers to one, in ice-cold 50ml polypropylene centrifuge tube, at ice by antibacterial Upper placement 10～20min, is centrifuged with 4000r/min with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) in 4 DEG C 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, uses with 10ml The 0.1mMCaCl of ice pre-cooling₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding turn Head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace of final residual Culture fluid flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, at this point it is possible to rapidly Cell being distributed into aliquot, freezes in liquid nitrogen ,-70 DEG C of storages are standby, hang from every kind of competent cell with the sterile pipette tip of cooling Liquid respectively takes 200 μ l and transfers in aseptic microcentrifugal tube, should add in often pipe DNA or coupled reaction mixture (volume≤ 10 μ l, DNA≤50ng), rotate gently to mix content, ice is placed 30min, centrifuge tube is put into pre-heating to 40 DEG C Circulator bath in test tube rack on, place 90s～2min, do not shake test tube, quickly transfer to pipe, in ice bath, make cell Cooling 1～2min, every centrifuge tube adds 800 μ lSOC culture medium, with water-bath, culture medium is warmed to 37 DEG C, is then transferred to by pipe On 37 DEG C of shaking tables, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, by appropriate bulk The competent cell that long-pending (each 90mm flat board is up to 200 μ l) have converted is transferred to containing 200mmol/LMgSO4 and corresponding antibiotic SOB culture medium on, flat board is placed in room temperature and is absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, may occur in which after 12～16h Bacterium colony.(ii) recon screening, expand and extract: select single colony inoculation in 5mL with sterile toothpick or disinfection inoculation pin In aseptic LB culture medium or rich medium (such as super broth or TB super broth culture medium), after overnight incubation, add In the 500mL 2L flask containing LB culture medium (containing suitable antibiotic), cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4, for Improving yield, surface area should be used relatively big and the flask of band deflection plate is to increase venting quality as far as possible, shaking speed should be greater than 400r/ Min), in 4 DEG C, 6000g is centrifuged 10min, by the 4mL resuspended precipitation of GTL solution, and transfers to the high speed of a volume >=20mL In centrifuge tube (bacterial precipitation can preserve at-20 DEG C or-70 DEG C of indefinite duration), add that 1mL newly joins containing 25mg/mL lysozyme GTE solution, resuspended precipitation, place 10min in room temperature, add 10mL and newly join NaOH/SDS solution, and gently mixing until liquid Body becomes homogeneous, limpid and thickness, places 10min on ice, adds 7.5mL acetic acid solution, is gently mixed until viscous with suction pipe Denseness declines and is formed big precipitation, places 10min on ice, and in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant To the centrifuge tube that another is clean, if there being visible drift that the isopropyl of 0.6 times of volume by several layers of filtered through gauze, can be added Alcohol, 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, and be vacuum dried (precipitation can be 4 DEG C of long-term preservations).(iii) recon Identifying: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence escherichia coli, ibid method is with restricted interior Cutting enzyme BamH I and carry out enzyme action, 10g/L agarose gel electrophoresis is identified, it is thus achieved that 2 bands of size about 2600bp and 5600bp, front Person meets the size of SV40T fragment in GenBank.

(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, 20% hyclone In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, Typically it is 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, from The heart, removes supernatant, standby.

(VI) importing of SV40T/pcDNA3.1 and amplification culture: prepare following solutions in 1.5ml microcentrifugal tube: pipe A, is dissolved in SV40T/pcDNA3.1 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 serum-free culture Liquid washs above-mentioned T lymphocyte 2 times.The training of 1ml serum-free is added in Lipofectamine-SV40T/pcDNA3.1 mixture Nutrient solution, mixes gently, then drops in above-mentioned T lymphocyte, and (hyclone concentration is to be subsequently adding 1ml serum-free medium 20ml/L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues Continuous cultivation 20h, discards culture fluid, and changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days select living cells After making amplification culture, then strengthen G418 concentration to 800mg L^-1, will can stablize the cell of growth in the G418 environment of high concentration Proceed amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If it is thin Born of the same parents increase slowly, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches Transferring in 75ml culture bottle during to 14ml, every 2-3 week adds 5-10ml fresh culture.Cell cultivates about 6-8 week the (the about the 55th Generation), still in logarithmic growth, i.e. incubation time and cell is accelerated in multiplication relation, and dead cell is less than 10% (by reading The scale taking culture vessel judges the increase situation of cell quantity；Dead cell and living cells is differentiated by trypan blue staining.Cause For normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not 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.Method be draw weekly a certain amount of Suspension culture, mix rearmounted room temperature 5～10 minutes with Trypan Blue agent, then make cell sheet, at microscope 1000 total cellular score of lower counting, calculate dead cell and the percentage ratio of non-staining living cells of coloring).Hereafter along with cultivating generation The increase of number and the prolongation of incubation time, the increase of cell quantity is slack-off, dead cell gets more and more, until cell no longer increases Add, even dissolve, reduce, all dead.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 minutes, Abandon supernatant, add 3ml freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, become cell (cell concentration is about 10 to suspension⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen In-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).

(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 and trains containing 15FBS low sugar DMEM culture medium Support bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation is until cell quantity is decreased obviously, every after cultivating 3 days The cell giving no count every 2 days changes liquid, uses same method to observe transfectional cell at hepato ZYME-SFM serum-free medium In growing state.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and makes 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 this cell is described System does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA colony in flow cytometry analysis cell line, as not having Have, illustrate that tumor feature does not occurs in cell line).Chromosome karyotype analysis method is: by adding preheating in 5mL culture fluid 250ug/ml Colchicine 100ul, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, fixing, film-making, G shows band post analysis karyotype；(iv) Flow cytometry: the cell ratio synthesizing, dividing in detection the 19th continuous cell line Example, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is the result that SV40 large T antigen is integrated, expressed. (viii) determined dna sequence: sequenator detection routinely, shows SV40 large T antigen DNA sequence.In (v) transfectional cell DNA SV40 big T gene test: as with Immunohistochemical detection, dye in the nucleus of SV40 transfection visible a large amount of brown particles, Show that SV40T antigen has been integrated into intracellular；Also T antigen expression in cell, wherein T antigen can be detected by RT-PCR method Primer: forward primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', downstream 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, expand body 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 With the cDNA of the 19th generation cell as template (carry out the synthesis of cDNA the first chain with reference to commercially available cDNA the first chain synthetic agent box, product- 20 DEG C of preservations)；Negative control sets two, does template with the cDNA of sterilized water, primary cell respectively, and positive control is with SV40DNA (extract SV40DNA with reference to SDS-proteinase-K pathway for template, because SV40 virus is without peplos, do not use SDS rupture of membranes, take 5 μ l and enter Row 1.5% agarose gel electrophoresis detects, and remaining-20 DEG C save backup)；(vi) mrna expression product measures: T antigen mRNA RT-PCR product checks order: take the amplified production of 100 μ l systems, reclaims test kit (Takara, Japan) with gel and reclaims product, takes 2 μ l DNA solutions dilute 100 times, survey 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 accords with after above-mentioned qualification Close immortalized cells characteristic the cell same or like with primary cell, take that growth conditions is good, be in exponential phase The cell of different generations, is performing centrifugal separation on (1 200r/min, 6min), resuspended carefully with the frozen stock solution 0.5～1ml containing dimethyl sulfoxide Born of the same parents, 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 is frozen, and 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 The molecule of function by conventional chemical coupling, crosslinking, affine absorption etc. be fixed on carrier make be coated with CD4 molecule Grain, or directly take the replacement CD4+ cell application of intimate granule.It is thin that the CD4+T cell of the present invention represents other CD4+ Born of the same parents, prepare immortalization CD4+T cell including with additive method.

Two, the preparation of acquired immune deficiency syndrome (AIDS) hemosorber

1, the filling of adsorbent

Take hybridoma macrophage strain and CD4+T cell strain prepared by the present invention, after cleaning with physiological saline solution, then with 1000r/min is centrifuged 5min (low speed is centrifuged in short-term), is 1: 0.5～3 by the ratio of hybridoma macrophage strain and CD4+T cell strain Ratio take sedimentation cell, assembling acrylate etc high-biocompatibility material (identical with plasma separator material) is made Cylindrical adsorption device, to 4/5, then adds cells frozen storing liquid (containing 30% hyclone, the RPMI-1640 of 12% dimethyl sulfoxide), Make cell concentration reach 80%, shake up gently, sealing, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen and freeze Deposit standby.When defrosting uses, need cryopreservation tube be put in 37 DEG C water-baths having been warmed up rapidly, and constantly shake, make in pipe Liquid melt rapidly, then with physiological saline solution clean after use.

2, the specification of adsorber

Prepared adsorber can be that footpath, the end is little, push up the cylinder that footpath is big, or square, infundibulate, volume be 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 (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, 2.5 mesh, 3.0 7 kinds of different sizes of mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh, in order to stop the inhibition of HIV of 120 nanometers or bigger thin Bacterium；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；Liquid Body is imported and exported and is provided with relief area between mesh screen, beneficially the stability of system circulation.

3, the material of adsorber

Hemosorber selects the macromolecular material of acrylate etc, it is desirable to good biocompatibility, activates benefit hardly Body, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can pass through covalency, The methods such as grafting, polymerization improve the structure of material, the regulation inhomogeneities on surface, hydrophilic, minimizing to blood coagulation and oxidative stress Impact thus improve biocompatibility, reduce complication generation.The material that some has anticoagulation is solidificated in carrier Or on the material of adsorber inner surface, blood coagulation can be suppressed, improve biocompatibility, also can reduce heparin consumption, and have can No-rod tractor can be realized.Heparin covalent is attached to polyether sulfone surface, the anticoagulation function of adsorber inner surface can be improved.At vinegar On acid fibrous membrane, covalent immobilisation linoleic acid film all can increase histocompatibility and anticoagulant effect.

Three, the preparation of separator

(1) preparation of blood separator

1, preparation principle: 1. hemocyte, antibacterial, the molecular size of virus: in blood of human body, visible component (cell) is big Little it is: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leukocyte is divided into 5 kinds, and neutrophilic granulocyte is about 12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, and small lymphocyte 6-8 μm, with erythrocyte Approximation, mononuclear cell is maximum, about 15-20 μm.Platelet is disc, diameter 1～4 microns to 7～8 microns, the blood of people Platelet average diameter is 2-4 micron, thick 0.5～1.5 micron.The size of antibacterial is: the diameter of coccus about 0.75-1.25 μm it Between, bacillus length is about in 2-5 μm, and spirillum is about 100-200 μm.Virus size with nanometer (nm) as unit [1cm= 10mm, 1mm=1000 μm, 1 μm=1000nm], between different virus, difference in size is very big, minimum such as the gemnivirus of plant (Geminiviruses) diameter only 18-20nm, maximum animal poxvirus (Poxviruses) size reach 300-450nm × 170-260nm, the longest as filamentous virus section (Filoviridae) virion size be 80nm × 790-14000nm, most The diameter of single virus particle is at about 100nm, and HIV (human immunodeficiency virus) is 100-120nm (0.1-0.12 μm).2. HIV sufferers blood Related compounds present in liquid: (gp120 with the CD4+ Cell binding of HIV cell surface is substantially for multinucleated giant cell Long-pending HIV cell), gp120 cell (surface is with the presence of gp120 but with the HIV cell of individual cells), gene integration Cell (integrate and have HIV double-stranded DNA, but the HIV that cell surface does not has gp120 is thin by HIV (human immunodeficiency virus) infection initial stage or incubation period Born of the same parents), normal white cell (being uninfected by granulocyte that the individual cells of HIV exists, mononuclear cell, lymphocyte), erythrocyte, blood little Plate, chemical analysis (protein, saccharide, lipid, electrolyte etc.), free HIV, antibacterial and other microorganisms.3. multinuclear is big and small Born of the same parents are natural large volume cell；Gp120 cell and gene integration cell can make cell by the immunoreation of antigen and antibody Coagulation is large volume many cells polymer；Free HIV can be changed into large volume composition by carrier granular/immunoreation.④ According to above-mentioned 3 points, can prepare can by individual cells but large volume cell or the blood separator of granule can not be passed through.5. select The material of the selective adsorption function of apparatus, screens out the HIV cell in blood through the extracorporeal circulation of blood branch road of the present invention.

2, the material of blood separator and requirement: with the adsorber of the present invention, selects poly-vinegar non-woven fabrics, acetate fiber, de- Fat is cotton, it is desirable to good biocompatibility, hardly activating complement, do not cause inflammatory reaction and leukocyte, platelet, blood oxygen to divide Pressure, the change of C3a, C5a.

3, the type and spec of blood separator: the profile of blood separator be prepared as column construction (with acetate fiber or Filter element made by the materials such as absorbent cotton), the shape such as flat structure (making filter element with materials such as poly-vinegar non-woven fabrics)；Aperture be prepared as 150～ 250 μm, 50～150 μm, 15～40 μm, 8～15 μm, 5～8 μm, 3～5 μm, 1～2 models such as μm.

4, the application of principle of blood separator: select the separator of different model, principle according to the state of an illness of HIV sufferers Upper first selection large aperture model does pre-sieving, the then model in selection of small aperture.Severe HIV sufferers often occurs serious Opportunistic infection, containing different size of composition in blood.As the fungus containing especially big volume, spirillum, tumor cell and His foreign body, then selecting aperture is 150～250 μm or the separator of 50～150 μm；As bitten carefully for the monokaryon of sieving HIV is huge Born of the same parents, multinucleated giant cell, many cells polymer and be adsorbed with the particulate matter of HIV, and in order to replace easily by HIV CD4+ cell, then select 15～40 μm, 8～15 μm, 5～8 μm, 3～5 models of μm etc.These several models approximate or are less than The volume of single erythrocyte, neutrophilic granulocyte, small lymphocyte in blood, but erythrocyte, neutrophilic granulocyte and macrophage tool There is the characteristic of amoeboid movement, can be by the micropore less than own vol.

(2) preparation of plasma separator

(1) preparation principle: prepare according to the molecular size of hemocyte and blood plasma components.Such as visible component in blood of human body The size of (hemocyte) is: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leukocyte is divided into 5 kinds, Neutrophilic granulocyte about 12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, small lymphocyte 6- 8 μm, approximate 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) material: can be selected for poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc., it is desirable to good biocompatibility, swash hardly Live complement, 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 be to blood coagulation and oxygen Change stress impact thus improve sieving adequacy and biocompatibility, the generation of minimizing complication.

(3) type and spec: for the profile of separator, can prepare as filter element with the material such as acetate fiber or absorbent cotton Become column construction, be prepared as the shapes such as flat structure with materials such as poly-vinegar non-woven fabrics as filter element；By hemocyte to be separated and blood The molecular size of slurry composition determines aperture.Plasma separator stable in properties involved in the present invention, good biocompatibility, penetrating Hollow fibre type filter made by the high molecular polymer that property is high, hollow-fiber film a diameter of 270～370 μm, and film thickness is 50 μm, Aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm.This hole is only permitted blood plasma and is filtered, but can stop that all of cell becomes Point.

Four, the component of AIDS immunoadsorption therapy instrument

1, key member: (1) blood separator: screen out with multinucleated giant cell or many cells polymer for by volume size The HIV cell that state exists, is i.e. used for removing endoglobar HIV；(2) plasma separator: be used for separating single blood thin Born of the same parents and blood plasma；(3) HIV adsorber: the HIV in adsorbed plasma.

2, additional member: include blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The part compositions such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring.(1) blood pump (Blood Pump): be used for promoting blood Circulating to maintain being smoothed out of blood purification treatment, usual blood pump part often has rotary test speed function, to monitor patient Blood circumstance, therefore blood pump runner and flute pitch set and want accurately and it needs to often adjust, according to the feelings of bloody path pump line Condition, is typically set as 3.2～3.3mm by spacing, can not be the most loose, and blood flow detection otherwise can be caused to be forbidden；Also can not be too tight, otherwise Pipe breakage can be caused.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump applied clinically, uses To continue injecting heparin in sieving pipeline (patient blood), owing to the blood of patient circulates and air contact, easily in vitro There is blood coagulation phenomenon, use heparin pump to be possible to prevent the generation of blood coagulation.(3) sound pulse pressure monitoring: arterial blood pressure monitoring mainly in order to The stopping state of dynamic monitoring blood separator micropore, additionally in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.When During blood flow deficiency, arterial pressure will reduce；When having blood coagulation, thrombosis, particularly during separator blockage of the micro orifice, arterial pressure will Raise；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 When there is blocking, venous pressure will raise.(4) air monitering (Air Detector): be used for monitoring the air gas of blood pathway Bubble, the principle of general ultrasonic listening, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble Time, detecting system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent the generation of danger.

In a word, on the basis of key member of the present invention and additional member, Import computer regulates and controls and makes the people of operation Property, the personalization for the treatment of, the safety of design, and modularity, automatically monitoring and regulation and control, liquid crystal display, judge voluntarily to warn The blood purifying therapeutical instrument that the report micro computer such as reason and ring off signal processes.

Five, the connecting path of AIDS immunoadsorption therapy instrument and using method

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

2, aerofluxus: with physiological saline solution topping up separator, adsorber and each circulation line, gets rid of separator, adsorber And gas in circulation line, bubble, go through, confirm without use after gas, bubble.

3, logical liquid: arterial blood line pipe (1) is connected the arteries of HIV sufferers, the row of going through the most again Gas is the most complete, and 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 (15) is connected venous blood Pipe, then opens blood pump (2), and blood flow is 100～150ml/min, such as Fig. 1, when arterial blood through arterial blood line pipe (1), When heparin and blood pump (2) enter blood separator (3), the large volume multinucleated giant cell formed because of HIV is delayed at In blood separator (3), mononuclear blood cell and blood plasma export (4), blood pump (6) and circulation line (7) through blood successively and flow into Plasma separator (8), the blood plasma of separation flows into now open adsorber (11) through blood plasma pump (9) and blood vessel (10) successively, Blood plasma to be full of, about 10 minutes, begin paying out blood plasma, through export pipeline (13) flow out, synchronize to adsorber (12) irrigate blood plasma, When blood plasma in adsorber (11) has nearly flowed, starting again at perfusion blood plasma, now adsorber (12) begins paying out blood plasma, and two The adsorber (11) of individual parallel connection and (12) are alternately.As represented Fig. 2 of the internal structure of the blood separator (3) in Fig. 1, blood There are a lot of micropore (3), multinucleated giant cell (4) micropore (3) can not be filtered and hindered on the tube wall of the inner chamber (2) of liquid/gas separator (1) Stay inner chamber (2), thus can be eliminated, can pass through in micropore (3), outside the mononuclear blood cell (5) of small size and blood plasma enters Chamber (6), then flows out through outlet (7), and then isolates hemocyte and blood plasma through the plasma separator (8) shown in Fig. 1.As represented Fig. 3 of the internal structure of the plasma separator (8) in Fig. 1, the tube wall of the inner chamber (2) of plasma separator (1) has a lot of micropore (3), it is impossible to export (8) by the mononuclear blood cell (4) of micropore (3) through the hemocyte with switchable valve and flow out, enter Fig. 1 Shown hemocyte export pipeline (14)；Plasma separator exocoel can be entered by the blood plasma of micropore (3) and chemical composition (5) thereof (6), then adsorber is entered through the blood plasma pump (9) shown in blood plasma flow export (7), Fig. 1 and blood vessel (10).As represented in Fig. 1 Adsorber (11) and Fig. 4 of (12) internal structure, when the blood plasma containing HIV (1) enters adsorber, HIV therein (1) is respectively It is thin that the macrophage (2) that is fixed in adsorber (6), CD4+T cell (4) are combined into macrophage phagocytic body (3), CD4+T Born of the same parents' conjugate (5), combined after HIV no longer move down, purify blood plasma through the outlet shown in Fig. 1 after adsorbed HIV The individual cells that road (13) separates with plasma separator (8) confluxes through venous line (15) after export pipeline (14) converges.As This purifies blood, removes HIV, until the plasma circulation amount being previously set (usually 9L), treatment just ends.Whole treatment Process is by computer control, and can detect duty, easy to use, automatization and safety at any time.

Six, the checking of AIDS immunoadsorption therapy instrument effect

1, blood separator filters the checking of HIV cell effect

The present inventor, according to the basic skills of the present invention, has done following simple confirmatory experiment: take Disease Control and Prevention Center and infection The some parts of anticoagulated whole blood of acquired immune deficiency syndrome (AIDS) (AIDS) patient made a definite diagnosis that sick laboratory biological Sample Storehouse preserves, respectively peek part phase It is mixed into 5 examples with the anticoagulated whole blood of abo blood group, makes blood flow volume sufficiently large, then entrust HC blood station, Zhejiang Province proportionately The blood component separation method of part blood transfusion, isolates leukocyte, erythrocyte, blood plasma through blood component piece-rate system, takes leukocyte Composition centrifugation routinely, inhales and abandons supernatant, with appropriate normal saline suspension leukocyte cell pellet, be subsequently adding proper ratio Gp120 antibody (Guang Rui bio tech ltd, Shanghai), mix rearmounted 37 DEG C react 5 minutes, then with aperture be 20～ The blood component piece-rate system of 30um isolates the leukocyte (the biggest leukocyte) of large volume, to the leukocyte filtrate filtered again The leukocyte (leukocyte in referred to as) of medium volume is isolated further with the blood component piece-rate system that aperture is 15～25um, Leukocyte in filtrate is the leukocyte (referred to as SL) of small size, collects large, medium and small leukocyte separation suspension respectively, Conventional centrifugal precipitates, and inhales and abandons supernatant, with the large, medium and small leukocyte cell pellet of quantitative liquid shifter draws equal amounts respectively, conventional method (machinery or cell pyrolysis liquid) cell lysis (as used lysate of the same race, need dosage equal), takes supernatant, then after centrifugation Operate according to HIV-1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai), with known dense The p24 antigen conduct of degree 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml Comparison, lowest detectable limit is less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, 15min Interior 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ml absorbance is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and testing result (table 1) is said Bright, the HIV-p24 content in the leukocyte of AIDS patient different volumes size is different, HIV-p24 in large, medium and small leukocyte Average content be respectively 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, in the most large and small leukocyte, HIV-p24 is average Content difference 148.6pg/ml, decreases 54.4%；In large, medium and small leukocyte, the total content of HIV-P24 is respectively 1375.0pg/ml, 979.9pg/ml, 632.1pg/ml, HIV-p24 total content difference 742.9pg/ in the most large and small leukocyte Ml, decreases 54.3%, through statistical test, t=2.43, p ＜ 0.05.Illustrate the large volume leukocyte in AIDS patient body or The large volume leukocyte formed after gp120 antibody effect contains the HIV of high level, can be by implementing the skill of the present invention Art scheme is separated removing.

HIV-p24 testing result (p24:pg/ml) in the large, medium and small leukocyte of table 1 AIDS patient peripheral blood

2, HIV adsorber (agent) removes the checking of HIV effect

(1) checking of CD4+T cell strain adsorption removal HIV effect

In order to verify effect of CD4+T cell strain adsorption removal HIV, the present invention devises easy method of testing: takes and goes out 2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5, draws by centrifugation CD4+T cell that (1000r/min, 5min) precipitate extremely respectively 200mm scale, then draws and is incubated after 100 DEG C dissolve at 39～41 DEG C of 0.9% standby agarose C1-4B, reach about The long scale of 10mm, 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 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 5 example blood plasma, the most about 10mL of patient, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube upper end blank pipe, erythrocyte sedimentation rate to be flowed through in batches The CD4+T cellular layer of pipe lower floor after flowing out in blood sedimentation tube, collects effluent, blood plasma after referred to as AIDS filter.Before taking AIDS filter Blood plasma after blood plasma and filter, according to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), the limited public affairs of biotechnology are inspired in Shanghai Department) 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, the range of linearity as comparison, lowest detectable limit In 0.5pg/ml～80pg/ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, is recognized as absorbance ＞ 0.12 For being positive, testing result (table 1) illustrates, after AIDS blood plasma filters the simple purifier containing CD4+T cell, part HIV is Being adsorbed by CD4+T cell, after the 1st time filters, HIV total body clearance is 22.84%, and after the 2nd time filters, total body clearance is 35.31%, after the 3rd time filters, total body clearance is 41.9%.Illustrate that HIV can be by the most clear along with the increase filtering number of times Remove, 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) checking of hybridoma macrophage strain adsorption removal HIV effect

For effect of check cross tumor macrophage strain adsorption removal HIV, the present invention devises easy method of testing: Take 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draw by centrifugation the macrophage that (1000r/min, 5min) precipitates respectively Hybridoma cell strain, to 200mm scale, is then drawn and is incubated after 100 DEG C dissolve at 39～41 DEG C of 0.9% standby agaroses C1-4B, reaches the long scale of about 10mm, and after putting blood sedimentation tube cooling, agarose becomes semi-solid, and blood sedimentation tube inner cell can be stoped to flow out But the material of water and the chemical analysis that will not stop little molecule etc passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse 5 example blood plasma, the most about 10mL of acquired immune deficiency syndrome (AIDS) (AIDS) patient preserved, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube in batches (simple purifier) upper end blank pipe, the hybridoma macrophage strain layer of blood sedimentation tube lower floor to be flowed through outflow in blood sedimentation tube After, collect effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, move suppression with human macrophage The factor (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) pairing detection, by specification operates, detection Scope is 0～800pg/ml, and sensitivity is 1.0pg/ml, directly can detect by an unaided eye: color in reacting hole under white background The deepest, the positive is the strongest, and negative reaction is colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also can survey OD value: on ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, surveys each hole after returning to zero with blank control wells 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 testing result in blood plasma before filter It is feminine gender (or preserving cause degraded etc. for a long time because content is less than detection sensitivity, blood plasma), and after filtering, in blood plasma, testing result is equal For the positive.Illustrate that macrophage hybridoma cell strain creates MIF cytokine in this process.MIF is collection cytokine, growth The factor, hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays The effect of central, plays panimmunity function in various infection and active chronic inflammation disease.Filter making AIDS blood plasma Before and after simple adsorber while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, resists according to HIV-1p24 Former detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operates, with concentration known 0pg/ml, 0.5pg/ The p24 antigen of ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, lowest detectable limit Less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, in 15min, 450nm measures extinction Degree (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ml extinction Degree is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and result (table 2) illustrates that AIDS blood plasma filters simple absorption After device, part HIV is by the phagocytosis absorption of macrophage hybridoma cell strain, and the blood plasma HIV after filtration significantly reduces, through the 1st After secondary filtration, HIV clearance rate is 20.55%, and after the 2nd time filters, HIV clearance rate is 42.83%, p ＜ 0.01, has substantially Effect, illustrate along with filter number of times increase HIV can constantly be removed, thus reach treat AIDS purpose.

Table 1 AIDS blood plasma filter MIF testing result before and after the Simple adsorption device containing hybridoma macrophage (quantitatively: pg/ml)

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

In a word, above-mentioned simple confirmatory experiment shows, is easily fused into large volume by the peripheral blood leucocyte of HIV many Core giant cell or many cells polymer, can be separated by blood separator and remove；And HIV free in blood plasma, can be by the present invention's Adsorbent (hybridoma macrophage strain, CD4+T cell strain) adsorption removal.Show with blood separator and HIV adsorber (agent) The AIDS immunoadsorption therapy instrument constituted for critical component has the significant treatment merit removing the inside and outside inhibition of HIV of blood cell Effect.

Claims (10)

1. the AIDS immunoadsorption therapy instrument for medical domain, it is characterised in that by made can be in conjunction with the hybridoma of HIV Macrophage strain and CD4+T cell strain are formulated in cells frozen storing liquid, and the exit that perfusion high-biocompatibility material is made is arranged The adsorber of screen cloth, makes adherent cell account for 4/5, and made adsorber collectively forms external suction with blood separator and plasma separator Adsorption device main part, for removing the multinucleated giant cell containing HIV and the HIV of blood plasma.

AIDS immunoadsorption therapy instrument the most according to claim 1, it is characterised in that the inner chamber of described blood separator leads to The micropore crossed on tube wall communicates with exocoel, and the aperture of described micropore is 1～250 μm, can pass through single blood cell but can not lead to Cross two and above mutual bonding or the large volume cell of fusion.

AIDS immunoadsorption therapy instrument the most according to claim 2, it is characterised in that the micropore hole of described blood separator Footpath is 150～250 μm, 50～150 μm, 15～40 μm, 8～15 μm, 5～8 μm, 3～5 μm, 1～2 μm.

4. according to the arbitrary described AIDS immunoadsorption therapy instrument of claim 1-3, it is characterised in that selection micropore size is 150～250 μm, 50～150 the separator of μm separate the fungus of especially big volume, spirillum and/or tumor cell；Select micropore hole Footpath is that 15～40 μm, 8～15 μm, 5～8 μm, 3～5 mononuclear phagocyte of separator separation HIV of μm, multinuclear are big and small Born of the same parents, many cells polymer, the particulate matter being adsorbed with HIV and/or the easy CD4+ cell by HIV.

AIDS immunoadsorption therapy instrument the most according to claim 1, it is characterised in that the hollow fibre of described plasma separator Dimension film a diameter of 270～370 μm, film thickness is 50 μm, and aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm.

AIDS immunoadsorption therapy instrument the most according to claim 1, it is characterised in that described adsorber is by its exit Screen cloth, be formulated in the CD4+T cell strain of agar gel, hybridoma macrophage strain collectively forms molecular sieve mechanical removal and cell The barrier of immune clearance blood plasma HIV.

7. according to the arbitrary described AIDS immunoadsorption therapy instrument of claim 1,6, it is characterised in that the volume of described adsorber Being 200～300ml, import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh, footpath sieve mesh at the bottom of exit Number is 2.0～5.0 mesh, and liquid outlet arranges the cell strainer that mesh number is 100 mesh, arranges rush between liquid entrance and screen cloth Enter the relief area of system stability circulation.

AIDS immunoadsorption therapy instrument the most according to claim 1, it is characterised in that described cells frozen storing liquid is containing 30% Hyclone, the RPMI-1640 of 12% dimethyl sulfoxide.

9. the preparation for the AIDS immunoadsorption therapy instrument adsorbent of medical domain, it is characterised in that with sterile physiological Saline cleans made hybridoma macrophage strain and CD4+T cell strain, and 1000r/min is centrifuged, and after cleaning, recentrifuge precipitation, presses The ratio that ratio is 1: 0.5～3 of hybridoma macrophage strain and CD4+T cell strain takes sedimentation cell, is formulated in cells frozen storing liquid, Load in the 200ml hydrostatic column that acrylate etc high-biocompatibility material is made, be fills up to 4/5, make HIV absorption Device, makes cell concentration reach 80%, shakes up gently, sealing, through 4 DEG C, and 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen Frozen standby, when defrosting uses, need cryopreservation tube be put in 37 DEG C water-baths having been warmed up rapidly, and constantly shake, make pipe In liquid melt rapidly, then with physiological saline solution clean after use.

10. claim 1-8 arbitrary described AIDS immunoadsorption therapy instrument application in preparing vitro Adsorption device, it is special Levy and be, described vitro Adsorption device include one end of arterial blood line pipe (1) through heparin and blood pump (2) with containing waste liquid outlet (5) blood separator (3) is connected, and blood separator (3) exports (4), blood pump (6), circulation line (7) and blood plasma through blood Separator (8) is connected, and the plasma outlet port of plasma separator (8) is through the absorption in parallel with two of blood plasma pump (9) and blood vessel (10) Device (11) is connected with adsorber (12), the export pipeline (13) of two adsorbers and the hemocyte outlet of plasma separator (8) Conflux through venous line (15) after converging in road (14).