CN106267413B

CN106267413B - AIDS plasma purification device

Info

Publication number: CN106267413B
Application number: CN201610539171.0A
Authority: CN
Inventors: 翁炳焕; 李兰娟; 钱欣; 刘蓓; 陈敏
Original assignee: 翁炳焕
Current assignee: Shu Lan (Hangzhou) Hospital Ltd.; Womens Hospital of Zhejiang University School of Medicine
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2019-03-29
Anticipated expiration: 2036-07-01
Also published as: CN106267413A

Abstract

A kind of AIDS plasma purification device for medical domain, it is characterized in that the separator of preparation energy separated plasma and haemocyte, former macrophage feature was not only retained with hybridoma technology preparation but also the hybridoma macrophage strain of energy indeterminate growth, building CD4+T cell strain is transfected using foreign gene and is expanded by growth stimulant of the peculiar molecular antibody in its surface, the cell strain of preparation is formulated in agar gel, clarifier is formed with high-biocompatibility material package, the layer distributed for the agarose concentration for forming gel from top to bottom from low to high, fixed macrophage strain therein and CD4+T cell strain play absorption HIV and are used, aperture for 1% agar gel of 85nm also there is the HIV of 100~120nm of detention to be used, made clarifier is applied in combination with separator and regulatory process, in extracorporal circulatory system Blood is divided into blood plasma and haemocyte by separator, and the purified device of blood plasma converges with haemocyte after filtering out HIV, then feeds back.

Description

AIDS plasma purification device

Technical field

The present invention relates to the preparations and application of AIDS plasma purification device in medical domain, are mainly used for AIDS patient's blood The removing of AIDS virus is starched, to achieve the purpose that treat AIDS.

Background technique

AIDS is passed caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV) It catches an illness, is widely current in the whole world, according to the related report of the World Health Organization (WHO) and The Joint Programme on AIDS, certainly Since U.S.'s discovery Patient With Aids cases in 1981, the whole world has 208 countries and regions to receive the serious of AIDS so far It threatens, there are about 40,000,000 people to have infected AIDS, and death toll is more than 20,000,000, and there are about 6000 people to become AIDS sense daily Dye person, while there are about more than 300 people to die of AIDS daily.It is in rapid growth period in HIV infected individuals in China, it is far super at present Cross 1,000,000.AIDS has become the great of after tumour, cardiovascular and cerebrovascular disease, tuberculosis, diabetes another facing mankind Infectious diseases, it has also become the serious public health of global concern and social concern.

Human immunodeficiency virus is a kind of slow virus (Lentivirus) for infecting human immune cells, belongs to reverse transcription disease One kind of poison, it is about 120 nanometers of diameter, substantially spherical in shape.Outer virionic membrane is lipoid coating (from host cell), and embedded with virus Albumen gp120 and gp41.Gp41 is transmembrane protein, and gp120 is located at surface, and with gp41 by noncovalent interaction in conjunction with.To It is inside the sphere matrix (Matrix) formed by albumen p17 and the half-cone capsid (Capsid) that albumen p24 is formed, capsid Under Electronic Speculum be in high electron density, include virulent rna gene group, enzyme (reverse transcriptase, integrase, protease) and other Ingredient from host cell.

It after HIV enters human body, is swallowed first by macrophage, but HIV changes certain positions in macrophage quickly Acidic environment creates the condition for being suitble to it to survive, is not killed not only and breeds in it instead.Because CD4 be HIV by Body, so the HIV bred in macrophage is through its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes itself hydrophobic effect mediate retroviral cyst membrane and cell membrane fusion) entering CD4+ cell, (cell, monokaryon macrophage are thin Born of the same parents, Dendritic Cells etc.), it is proliferated rapidly in the cell, generates 10 daily⁹~10¹⁰Virion, and it is normal constantly to enter other And regenerated CD4+ replicate into the cell, manufacture more virus infected cells, make peripheral blood CD4+T cell sustaining breakdown, subtract It is few.The T cell of infected Apoptosis or even infected by HIV can be directly activated to express in conjunction with gp120 and the CD4 receptor of HIV Envelope antigen can also start normal T-cell, cause a large amount of broken of CD4+ cell indirectly by cell surface CD4 molecule cross-link Bad, as a result cause the severe immune deficiency centered on CD4+T cell defect, patient mainly shows: periphery lymphocyte is reduced, T4/T8 proportional arrangement disappears to the reaction of phytohemagglutin phytolectin and certain antigens, delayed allergy decline, NK cell, macrophage Cell activity weakens, and the synthesis of the cell factors such as IL2, interferon is reduced.CD4+T cell is most important immunocyte, infection Person once loses a large amount of CD4+T cells, and entire immune system will all lose the infection of various diseases by deathblow Go resistance.HIV can also show as hiding without showing clinical symptoms, genome for a long time after entering host's CD4+ cell RNA reverse transcription enters in host cell nuclear at double-stranded DNA with viral integrase enzyme, under the action of integrase, double-stranded DNA integration Into host cell gene group, the viral DNA being integrated is known as provirus, and the several months that can hide does not replicate even for many years, causes The incubation period of AIDS several months to many years.In the incubation period of AIDS, HIV is mainly in the macrophage and Dendritic Cells of lymph node Breeding, these cells are intracorporal HIV depots, and releasably the CD4+T cell into peripheral blood or transfection peripheral blood, there is silk point Splitting original, antigen, TNF, IL-2 and lymph plain (LT) can excite HIV provirus gene multiple in the CD4+T Intracellular transcription of infection System.After being largely proliferated, inhibition of HIV particle constantly discharges from the infection cell being destroyed and is free on blood, then enters back into Other cells continue course of infection.

Body production envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody can be stimulated after HIV infection.? Low-level antiviral neutralizing antibody is measured in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers' highest illustrates that the antibody has protective effect in vivo.But antibody cannot be with the virus that retains in mononuclear macrophage Contact, and antigenic variation easily occurs for HIV envelope protein, original antibody is ineffective, keeps neutralizing antibody due from playing Effect.In the latent infection stage, HIV provirus is integrated into host cell gene group, therefore HIV will not be known by immune system Not, so relying solely on itself immune function can not be removed.Another critically important reason should be killed according to antibody It goes out, remove the mechanism speculate of antigen, after immune antibody and antigen binding, if to generate immunological effect or pass through activation Complement mediates ADCC effect to dissolve cellular antigen, but HIV is not cellular antigen；Attracted by chemotaxis and is swallowed Antigen is removed in cell phagocytosis, but HIV is protected in phagocyte instead, is proliferated；Antibody and antigen binding, which rise to neutralize, to be made With being allowed to lose appeal, but HIV antigenic structure is changeable, is often difficult to antibody.

In terms of the treating AIDS method for having been used for clinic at present, effect is less ideal: (1) hiv reverse transcriptase inhibits Agent: being only capable of preventing the permissive cell of not yet infected by HIV from infecting, and does not have a therapeutic effect to the cell infected, and toxic side effect compared with It is more, including gland plastochondria toxicity, bone marrow suppression, globular anemia, granulocyte and decrease of platelet, pancreatitis, intersect in addition resistance to The generation of pharmacological property, this kind of medicine are not used alone, mainly do drug combination, and are also easy to produce drug resistance mutant, cause under clinical efficacy Drop or failure.(2) toxic side effects such as drug induced hepatic injury, disorders of lipid metabolism and drug resistance hiv protease inhibitor: are also easy to produce Property.(3) it hiv integrase inhibitor: by inhibiting inhibition of HIV DNA to play a role with host cell DNA integration, is reversed with HIV Transcriptase inhibitors and protease inhibitors are combined while use has synergistic effect to antiviral.(4) inhibition of HIV is inhibited to enter inhibition Agent: including block gp120 with CD4 ining conjunction with, blocking HIV in conjunction with accessory receptor, act on gp41 film subunit and act on T drench Bar cell surface CC-chemokine receptor 5 (CCR5) has side effect to liver and heart to block HIV to enter host cell.(5) Cytokine therapy: it is mainly used for regulatory T-cell dynamic equilibrium.(6) HIV vaccine is treated: such as intrinsic due to the particularity of HIV Immune to be not enough to resist HIV and its targeting destruction immune system, virus mutation is rapid, causes not yet to develop real safety so far Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, including antisense technology, RNA bait, RNA dry It disturbs, intrabody, dominant negative mutant, suicide gene etc., but enters the gene therapy in II clinical trial phase stage almost No.(8) monoclonal antibody passive immunization therapy: the neurological susceptibility of HIV is reduced by lowering CD4+T cell surface CCR5, is prolonged The progress of slow AIDS and the diffusion for reducing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10, which are applied to HIV infection person, to be had Good tolerance and safety can delay but cannot prevent rebound (9) adoptive immunity cell therapy of virus: external a large amount of It will lead to viral massive amplification when the culture self CD4+T cell of HIV, increase the CD4+T cell quantity of virus infection, and feed back CD4+T cell may will increase the place of internal virus replication, lead to virus load bounce-back, and on the whole, adoptive immunity is thin Born of the same parents treat without apparent toxic side effect, 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 one kind of T lymphocyte, average Service life is generally 7 days or so, but certain T cells can long-term surviving, unlimited amplification especially after immortality is melted into cell line (strain). Foreign literature report, simian virus 40 (SV40) can be such that certain human cells immortalize.Poulin DL, Kung AL and Sullivan CS etc. is studies have shown that the importing of SV40T antigen gene can accelerate the growth rate of transformed cells, immortalized cells Repeatedly still there is metastable multiplication characteristic and functional status, while can also retain its initial cell after passage in vitro Many phenotypic differentiations.Reilly establishes vascular smooth muscle cells strain with the genetic transformation of simian virus large T antigen, constructs cell membrane Type is to study the inhibiting effect mechanism of heparin for vascular smooth muscle.Su etc. utilizes the superficial cell strain converted through SV40, structure Cell model is built to analyze the regulating and controlling effect of epithelial cell internal protein synthesis.Miquel etc. is thin with the cuticulated epithelium that SV40 is converted Born of the same parents' strain, the cell adhesion mediated as cell model research laminin 5.The forefront converted through SV40 such as Webber The physiological function and secreting function of prostate epithelial cell are studied in glandular epithelium strain as cell model.Racusen etc. is used Renal cells model is converted through Ad12-SV40 to study the damage and disease of proximal convoluted tubule.Hougton etc. is turned with SV40 Change establishes Bone marrow Stromal cell as cell model to study under certain condition of culture, cell with to fat cell and at The potential of the two-way differentiation of osteocyte further studies the mechanism of osteoporosis.Foreign study, which is also shown that, imports exogenous human end Human telomerase reverse transcriptase (hTERT) can make cell keep normal phenotype and differentiating characteristic.It is successfully established using hTERT in recent years The immortalized cell lines of certain cells, substantially holding chromosome stabilityX, differentiation be normal, contact inhibition, opposite without oncogenicity etc. Normal growth characteristics.In dentistry field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establish immortality Change people's Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number up to 150 times or more, Cell shows original biological characteristics, and the GAP-associated protein GAP of derived cell can be expressed after Fiber differentiation.Kitagawa etc. Transfection hTERT establishes people cementoblast system, and cell multiplication is up to 200 times or more, cell differentiation marker such as alkaline phosphatase The expression such as enzyme, type i collagen are stablized.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, brain edema cell model etc..So CD4+ can be prepared T cell strain is used to prepare clarifier after mass propgation, with the HIV in CD4+T cell clearance blood plasma.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophil leucocyte in peripheral blood, macrophagocyte It is the macrophage in the monocyte and a variety of organs, tissue in blood, the two constitutes mononuclear phagocyte system.Monocyte It is formed by the monocyte precursor Development And Differentiation in marrow, accounts for about the 3%-8% of blood middle leukocytes sum, volume is compared with lymph Cell is bigger, and monocyte only stops 12-24 hours in blood, and subsequently into connective tissue or organ, it is huge for reaching maturity Phagocyte, macrophage are highly differentiation, mature cell type in mononuclear phagocyte system, have stronger phagocytosis function Can, wandering macrophage is greater than monocyte several times, and it lasts a long time, can survive in the tissue some months, the macrophage of colonization There is different titles, be Kupffer Cell in liver, be in brain microglia, be osteoclast etc. in bone, expresses Fc Receptor, C3b receptor and CD14 play defense function in inherent immunity, and the professional antigen of participation adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of AIDS virus (HIV), thin by macrophage first after HIV enters human body The phagocytosis of born of the same parents, but HIV changes the acidic environment at certain positions in macrophage quickly, creates the condition for being suitble to it to survive, It is not killed mass propagation aggregation in it instead not only, then HIV is passed into CD4+T cell.So can be with conventional hybridization Oncocyte technology of preparing, prepares macrophage hybridoma, and the clarifier for the treatment of AIDS is used to prepare after massive amplification, The HIV in blood plasma is removed with the phagocytic function of macrophage.

Agar Gel or agarose are a kind of polysaccharide bodies containing sulfate, and when high temperature can be dissolved in water, are congealed into after cooling solidifying Glue forms a kind of porous reticular structure, allows macromolecular substances (molecular weight is up to million or more) to pass freely through, aperture Size put to death in agar concentration, concentration is bigger, and aperture is relatively small, and concentration is smaller, and aperture is relatively large, 1% agar gel Aperture be about 85nm.Since agar or agarose have good chemical stability, water content is big after gel, and transparency is good, Convenient sources, it is easy to handle, it is a kind of good dispersive medium, is with by the immunodiffusion of dispersive medium of agar gel Know the routine experiment checkup item of the corresponding unknown antigen of antibody test, and in " Chinese Pharmacopoeia " 2010 editions regulation for influenza disease The standard method of malicious vaccine hemagglutinin content detection.When a kind of solution passes through semi-solid gel, macromolecular solute therein is just The gel pore detention acted on by molecular sieve is in gel, when patient flows through gel through the blood plasma that extracorporal circulatory system is isolated, blood plasma In 100~120nm HIV can be about by aperture 85nm 1% concentration agar gel detention in gel, thus can remove blood HIV in slurry.

In short, various drugs and biological products can not effectively kill intracorporal AIDS virus, and price, side effect is big, So far the effective ways for the treatment of AIDS be there is no, it has also become attack the global problem being unable to long.

Summary of the invention

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

The invention aims to provide AIDS plasma purification device；Another object is to provide for AIDS plasma purification device Preparation and application method.

The object of the present invention is achieved like this: the separator of preparation energy washed corpuscles and blood plasma is turned with foreign gene The method building CD4+T cell strain of dye is simultaneously expanded using the peculiar molecular antibody in its surface as growth stimulant, with hybridoma technology Preparation had not only retained former macrophage feature but also the hybridoma macrophage strain of energy indeterminate growth expands parallel, took CD4+T cell strain With macrophage strain with high-biocompatibility material package and prepare can prevent cell and its fragment from filtering out and can gulp down for cell strain Bite absorption HIV provide place plasma purification device, with rise carrier function after 100 DEG C dissolve keep the temperature 39~42 DEG C 0.7 The agarose of~1.1% gradient concentration sequentially adds plasma purification device from high to low by concentration, after being cooled to semi-solid gel Followed by the layer distributed for making the gel in clarifier form agarose concentration from low to high from top to bottom next time is added, have Conducive to the effect of plasma perfusion and molecular sieve and immune clearance, wherein CD4+T cell strain and macrophage strain are fixed on fine jade In sepharose, play a part of to adsorb HIV, prepared clarifier is applied in combination with separator and regulatory process, extracorporal circulatory system In blood blood plasma and haemocyte are divided by separator, converge after the purified device absorption HIV of blood plasma with haemocyte, then feed back.

Technological core of the invention is made of plasma separator and clarifier, and wherein plasma separator is used for washed corpuscles And blood plasma, the cleanser in clarifier is made of the CD4+T cell strain and macrophage strain for being fixed on agar gel, because of CD4+T The CD4 molecule of cell surface is the receptor of HIV and becomes the permissive cell of HIV, and HIV can be adsorbed when meeting with HIV, is adsorbed HIV is fixed in agar gel with CD4+T cell, because of the natural phagocytosis characteristic of hybridoma macrophage, energy when HIV meets therewith It is fixed in agar gel therewith by phagocytosis, and agar gel is formed by filter opening and subtracts with increasing for agarose concentration Few, clarifier entrance agarose concentration is low, and filter opening is just big, is conducive to plasma perfusion and cell and the association reaction of HIV；And go out Concentration is high at mouthful, and filter opening is just small, is easy to detention HIV or Large molecular conjugates, and clarifier is combined with a variety of special and non-specific HIV removes composition, in order to avoid extraordinary strain is because of the futile treatment caused by immunity difference, so in vitro in circulation blood by separator After being divided into blood plasma and haemocyte, blood plasma flows through when clarifier HIV therein because by CD4+T cell and the absorption of hybridoma macrophage And removed by agar gel detention, purified blood plasma is fed back after converging with haemocyte, and the present invention is with external mechanical removal The method substitution of HIV can not effectively kill the conventional anti-reverse transcription drug treatment in vivo of HIV for a long time, realize artificial By HIV from the treatment new method of internal mechanical removal.

Specific embodiment

Fig. 1 is the application schematic diagram of the AIDS plasma purification device proposed according to the present invention.

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

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

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump (3) it is connected with plasma separator (4), plasma separator (4) purification in parallel with two through blood plasma pump (6) and circulation line (7) Device (8), clarifier (9) be connected, be then successively connected with circulation line (10), venous line (5), venous line (5) it is another End is connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 be plasma separator inner cavity, and 3 be the micropore on the tube wall of plasma separator inner cavity, 4 Being cannot be by the haemocyte of micropore (3), and 5 be the blood plasma and chemical analysis that can pass through micropore (3), and 6 be plasma separator exocoel, 7 be blood plasma outflux, and 8 be that there is the haemocyte of switchable valve to export.

In Fig. 3,1 is free HIV, and 2,4 be respectively the hybridoma macrophage strain being fixed in agar gel (6), CD4 + T cell, 3,5 be respectively to be delayed in agar gel (6) after HIV and the strain of hybridoma macrophage, CD4+T cell combination Conjugate, 7 is by the large volume of HIV of agar gel (6) molecular sieve detention.

Below with reference to Fig. 1, Fig. 2 and Fig. 3, the embodiment of AIDS plasma purification device proposed by the present invention is made detailed Description.

One, the preparation of 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 centrifugation method and monokaryon macrophage Cell.Specific method is: the Cord blood buying the White Blood Cells Concentrate of Blood Center or saving for scientific research takes 2mL sample, PBS 6mL anticoagulation is slowly superimposed on dropper that 4mL lymph has been added is thin by 2~3 times of hemodilution, after mixing well by liquid along tube wall Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tube of born of the same parents' separating liquid；It is divided into pipe after centrifugation 3 layers, upper layer is blood plasma and PBS liquid, and lower layer is mainly red blood cell and granulocyte, and middle layer is lymphocyte separation medium, in upper, middle layer It is PBMC that, which there be the white cloud and mist layer narrow band based on mononuclearcell in interface, is inserted into cloud and mist layer with capillary syring, is drawn PBMC is placed in another 50mL centrifuge tube, is added 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant Cell is resuspended in 50mLPBS, is centrifuged (350r/min, 20 DEG C) 15min, abandons supernatant, and Buffer (PBS+0.5% new life ox blood is added + 2mmol/LEDTA clearly, pH7.2) 2mL resuspension cell, it takes 15uL cell suspension to be added on blood counting chamber and counts 4 under microscope Cell (PBMC) sum in a block plaid.

(2) it single core histocyte: is provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, preparation method are: the 1. acquisition and transhipment of spleen tissue: in the approval of the reason committee and patient's informed consent Under, the spleen sample tissue for taking operation to cut off shreds into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to cell culture chamber rapidly.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS are washed 3 times, and RPMI-1640 is washed 2 times, to remove the blood in tissue and guarantee the sterile of tissue.Machine Tool grinds spleen tissue, at this moment just has a large amount of histocyte is outstanding to be mixed in RPMI-1640 liquid.With 200 mesh stainless steel filtering net mistakes Filter is outstanding to be mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing red blood cell, lymphocyte, Macrophage etc.).3. the cracking of red blood cell in spleen tissue cell suspension: and then centrifugation is washed with RPMI-1640 liquid (1000r/min, 3min) is added Tris-NH4Cl and acts on 5min, splitting erythrocyte, Quick spin to remove cell debris (1000r/min, 3min), remove supernatant in splitting erythrocyte fragment, PBS washing centrifugation 3 times, RPMI-1640 wash from The heart 1 time, to remove Tris-NH4Cl remaining in suspension, it is avoided to influence the survival of cell, at this point, mainly containing in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as culture Cell stoste, Trypan Blue determine vigor and count, and are (3~5) × 10 with RPMI-1640 liquid adjustment cell concentration⁶/ L, will The cell suspension inoculation of concentration is adjusted in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2~3h, observe form under phase contrast microscope.The digestion of adherent spleen tissue macrophage: adherent spleen tissue macrophage The digestion of cell: sucking culture supernatant, and macrophage is adherent, and PBS blows and beats repeatedly, digests, the washing centrifugation of gained cell suspension (1000r/min, 3min), the macrophage isolated and purified.Further, it is also possible to which the sample discarded after treatment or operation is taken to mention Take preparation, such as cavum peritoneale liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa.

(3) amniotic fluid, villus cell: Zhejiang University's attached hospital for obstetrics and gynaecology's reproduction heredity laboratory is spare.In reason committee member Can ratify under patient's informed consent, take laboratory diagnosis report after remaining amniotic fluid, villus cell, select logarithmic growth phase cell Continue to employ.

2, cell culture and the adherent preliminary sorting of macrophage

Routinely cell culture, but according to the difference of cellularity, appropriate adjustment incubation time, condition of culture etc., generally Single core blood cell (PBMC) or single core histocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method Culture dish in, in 37 DEG C, 5%CO₂Cell incubator (Themo electro corporation CLASS 100, beauty State) in be incubated for 2h, after mononuclearcell is adherent, inhale abandon upper layer suspension cell (cell other than macrophage be not easy it is adherent and Removed with upper liquid), PBs buffer gently washs 3 times, and a small amount of RPMI-1640 culture medium is added, is scraped with cell scraper adherent Cell (predominantly macrophage, but there are also other a small amount of attached cells).1 000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid is thin There is cell growth clone in born of the same parents, villus cell culture 1~7 day, cell growth converges the logarithmic growth phase that rate reaches 60~80% Cell is digested with pancreatin, and PBS cleaning obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorts

Sort cd4 cell: 1. main agents and instrument using immunomagnetic beads method: (German U.S.A day Ni is biological for CD4 immunomagnetic beads Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；Newborn bovine serum (Hyclone company)；MiniMACS magnetic separation system (German Mei Tian Ni Bioisystech Co., Ltd).2. cd4 cell is immune Magnetic bead sorting method: cell suspension, which is divided equally to two 1.5mLEppendorf, manages, and is centrifuged (300r/min, 20 DEG C) 10min, discards Supernatant is resuspended the every 80uLBuffer of cell and contains cell number 10⁷It is a, every 10⁷A cell add 20uLCD4MicroBeads or CD8MicroBeads is mixed well, and in 4~8 DEG C of hatching 15min, washs cell with 1mLBuffer, be centrifuged (300r/min, 20 DEG C) 10min, it discards supernatant 500uLBuffer and cell is resuspended, MS splitter is placed in the magnetic field of MACS separator, with 500uLBuffer rinsing is rinsed splitter repetitive operation 3 times by 500uL cell suspension by splitter with 500uLBuffer, Efflux is collected, contains non-cd4 cell in efflux, splitter is taken out from separator, with 1000uLBuffer pressure flush point From column, collection efflux, for cd4 cell, (cell viability detection: taking 15uL cell suspension respectively before and after cell purification and waits bodies for this Product trypan blue solution mixing, the not colored shinny person of microscopically observation are living cells, and the coloring person of swelling is dead cell, calculate 200 The percentage of living cells in a cell).The cell sorted at this time is mainly macrophage.

4, CD14 cell (macrophage) sorts

CD14 is monocyte and the distinctive surface marker of macrophage, theoretically if from single core histocyte, sheep It is sorted in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, gained Cell includes monocyte and macrophage；But because the monocyte service life is short, only survive 1 day in peripheral blood and can not show a candle to macrophage Cell is easy to adherent growth, so removing substantially in cell adhere-wall culture of the invention, the cell sorted out is essentially Macrophage.

Basic skills is analogous to cd4 cell, using immunomagnetic beads method.1. reagent: people's CD14 immunomagnetic beads kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead and spy Anisotropic one monocyte of target cell combines: every 1 × 10⁸The magnetic bead and 800uL buffering of 200uL coupling CD14 antibody is added in a PBMC Liquid (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/L EDTA0.5mL, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube It mixes well, 4 DEG C of incubation 15min, centre can slightly shake 1 time.Take out centrifuge tube after 15min, every 1 × 10⁷A cell is added 1 Buffer is pre-chilled in~2mL, and 1 000r/min is centrifuged 8min, abandons supernatant, and 0.5mL buffer is added and blows and beats into single cell suspension. (B) it collects the monocyte of marked by magnetic bead: cell splitter is placed on MACS magnetic frame, 1mL buffer statocyte is added Splitter drips to no liquid, immediately adds above-mentioned cell suspension in people's cell splitter, rinses cell with 0.5mL buffer Splitter 3 times.After to be rinsed, 1mL buffer is added, the emigrated cells splitter from magnetic frame is quickly pushed with needle column, Go out the cell combined in splitter with one magnetic bead of CD14 antibody, the as macrophage of CD14+.

In addition, following 2 kinds of methods sorting, including 1. adherent method also can be used: PBMC being placed in and is cultivated containing RPMI-1640 In the culture dish of base, in 37 DEG C, contain 5%C0: cell incubator (Themo electro corporation CLASS 100, The U.S.) in be incubated for 2h.It after adherent mononuclear cells, inhales and abandons upper layer suspension cell, PBs buffer gently washs 3 times, is added 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: CD14 label: taking PBMC, is adjusted with buffer (bovine serum albumin(BSA) 2.5mL and the 2mol/LEDTA 0.5mL containing 10%) Whole cell density is 1 × 10⁸CD14+-FITC antibody 100uL is added in/mL in every milliliter of cell suspension, and 4 DEG C are protected from light label 18min, then 1mL streaming buffer is added to terminate dyeing into centrifuge tube, PBs is washed 3 times, with the PBS for containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation in flow cytometer (BD FAcsAria II, the U.S.) on sort, according to the fluorescence intensity of CD14 antibody, the relative particle of the relative size of cell and cell and interior The complexity of portion's structure collects the cell of CD14+.

5, prepared by CD14 hybridoma cell strain (strain of hybridoma macrophage)

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire ox Serum (FBS) purchases Jinshi City on daytime ocean Hao biological products science and technology responsibility Co., Ltd；DMSO (- methyl sulfoxide) is the pure examination of domestic analysis Agent.

(2) myeloma cell prepares: fusion the last week takes out the myeloma cell (SP2/ that a pipe freezes out of liquid nitrogen container 0), be immediately placed in hot water thaw (using it is most be Sp2/0 cell strain, the cell strain growth and fusion efficiencies are good, itself is not Any heavy chain immunoglobulin or light chain are secreted, the highest growth scale of cell is 9 × 10⁵/ ml, the doubling time is usually 10~ 15h；Selection homologous cell strain is considered in practical application relevant to human body, if Shanghai Fu Xiang Biotechnology Co., Ltd is to ATCC The NCI-H929 human myeloma cell strain that cell bank is introduced).Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min； It is repeated 1 times.Sediment is moved into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, once passed within 3-4 days In generation, expands culture, and fusion adjusts cell state in first 24 hours, guarantees that cellular morphology is good before merging, growth is vigorous.It is added Appropriate basal medium gently beats 1000r/m centrifugation 5-10min after mixing, washes repeatedly cell 2 times into centrifuge tube.

(3) CD14 cell (macrophage) to be hybridized prepares: the mononuclear macrophage that the present invention sorts is with basal medium Total cell number is adjusted to 1 × 10⁸~2 × 10⁸For cell fusion.Blue dyeing phase-contrast microscopy, viable count are expected with platform It should be higher than that 80% is qualified.

(4) cell fusion: CD14 cell (mononuclear macrophage) and myeloma cell are added with 10: 1-5: 1 ratio In centrifuge tube, it is mixed evenly, 1000r/m is centrifuged 5min, discards supernatant, and it gently beats tube bottom to cell grainless and precipitates, weight It is 2 times multiple.Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up PEG3000 is added drop-wise in fusion pipe in 60s along tube wall while gently rotating centrifugal pipe, later trains the basis of the 25mL of preheating It supports base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal pipe during addition is then allowed to stand In 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T culture medium is added.It is inoculated into after appropriate mixing In 96 well culture plates, 37 DEG C are placed in, is cultivated in 5% CO2 incubator.

(5) the mononuclear macrophage strain with phagocytic function is screened: cell growth status in 96 well culture plates of observation, 7-10 Division can be grown by only having hybridoma after it, discarded HAT culture medium at this time, replaced complete medium.Cell clone growth When area reaches 1/10 cell hole, culture supernatant is gone, selection has the culture hole of the good hybridoma cell strain of growth conditions, shows Position, the size that cell strain growth is marked under micro mirror, draw cell clone in the position of mark using sterile pipette tips has to new In the culture hole of complete medium, then successively doubling dilution to hole is counted below, and 37 DEG C, 5%CO2 incubator is interior to cultivate a Zhou Zuo The right side, microscopically observation cell growth status take in cell or culture when cell clone is covered with to 1/10 or more hole floor space Clear detection hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain swallows bacterium Function detection: macrophage and staphylococcus or Candida albicans are hanged Liquid mixing incubates, and smear is fixed, the dyeing of serge blue liquid, in oily phagocytosis situation under the microscope, counts phagocytosis bacterium and does not gulp down The number of macrophages ratio of bacterium is bitten, to swallow the strong macrophage of bacterium function alternately positive clone strain.

2. hybridoma macrophage strain swallows HIV Function detection: AIDS (AIDS) patient's for taking Disease Control and Prevention Center to save After blood plasma and hybridoma macrophage strain mixed culture, cell strain is separated, PBS is cleaned 3 times, measures the phagocyte strain through cracking The function of swallowing HIV, with specific reference to HIV-1p24 antigen detection kit, (enzyme-linked immunization, Shanghai inspire biotechnology limited Company) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is lower than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity as control, minimum detection limit 450nm measures absorbance (OD) in 0.5pg/ml~80pg/ml, 15min, and blank control calibration object absorbance value is not higher than 0.050,0pg absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbance, is recognized as absorbance > 0.12 To be positive.Specifically operated by kit specification.

3. hybridoma macrophage strain generates macrophage cytokines detection: with human macrophage migration inhibitory factor (MIF) The operation of ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) by specification, detection range are 0~800pg/ml, Susceptibility is 1.0pg/ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive stronger, negative To be colourless or extremely shallow, the depth of the be in color of foundation is indicated with "+", "-" number for reaction.OD value can also be surveyed: in ELISA detector On, at 450nm (if developing the color with ABTS, 410nm), each hole OD value is surveyed after returning to zero with blank control wells, if more than defined 2.1 times of negative control OD value, it is as positive, specifically operated by kit specification.MIF be collection cell factor, growth factor, The multi-effect protein molecular of hormone and enzyme characteristic plays central as inherent immunity and the regulatory factor of inflammatory reaction Effect, it is various infection and active chronic inflammation disease in play panimmunity function.

According to testing result, select the cell clone in the culture hole with stronger macrophage function repeat it is next Wheel dilution culture, repeats 2-3 wheel, and detection function is taken out after stablizing, and is transferred to culture bottle mass propgation.

(6) preservation and recovery of hybridoma macrophage strain: preceding 12 hour adjustment cell growth state is saved, one bottle of life is taken Long vigorous, the good cell of form, is made cell suspension after appropriate digestion, 1000r/min is centrifuged 5min, removes supernatant, flick Tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and dispenses cell cryopreservation tube, and 1mL/ is managed, and -70 Cryopreservation tube, is put into liquid nitrogen container after taking-up and saves backup by DEG C refrigerator overnight.40 DEG C or so of hot water is got out before recovery, it will Cryopreservation tube carefully takes out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, and is centrifuged after defrosting in 1000r/min 5min opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting washed once with complete culture solution, then It is centrifuged 5min in 1000r/min, is discarded supernatant, in case making to expand culture.

6, hybridoma macrophage strain treatment cell preparation

That is the amplification cultivation of hybridoma macrophage strain.Above-mentioned cell precipitation is gently resuspended using complete culture solution and is moved back Enter in culture bottle, sets 37 DEG C, cultivated in 5%CO2 incubator.Amplification cultivation is passed on repeatedly, until required hybridoma cell strain In quantity, every 10 generation of secondary culture positive hybridoma cell strain, detect the function of macrophage hybridoma cell strain, see whether steady It is fixed.Continuation carries out extensive industrialization preparation in several bottles, saves backup.

(2) preparation of CD4+T cell strain

1, the source of primary lymphocyte

Have with sow by way of: 1. for scientific research save Infectious Diseases Lab sample database in freeze lymphocyte strain (warp Inactivation HIV totivirus is immune but is uninfected by the lymphocyte of HIV)；2. buying the fresh White Blood Cells Concentrate in blood station, then went out The immune lymphocyte of HIV infection strain living；3. the T lymphocyte system (strain) directly bought from businessman；4. being saved for scientific research Cord blood lymphocytes cell (immune through inactivation HIV)；5. being directly derived from the peripheral blood lymphocytes of HIV-1 the infected (for certainly Body), mononuclearcell (PBMC) is separated using Histopaque lymphocyte separation medium.

2, the preparation of CD4+T cell

1. main agents and instrument: CD4, CD8 immunomagnetic beads (German Mei Tian Ni Bioisystech Co., Ltd)；Isothiocyanic acid Fluorescein CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company)；(perseverance letter in Shanghai is biochemical for lymphocyte separation medium Reagent Co., Ltd)；(the raw work biotechnology service in Shanghai is public for ethylenediamine tetra-acetic acid (EDTA), 0.2% Trypan Blue liquid Department)；Newborn bovine serum (Hyclone company)；MiniMACS magnetic separation system (German Mei Tian Ni Bioisystech Co., Ltd)； EpicsXL type flow cytometer (BeckmanCoulter company, the U.S.).2. separation (the density gradient of mononuclearcell (PBMC) Centrifugal process): it is sterile to take 20mL blood sample, and heparin sodium (500IU/mL) 2mL is anticoagulant；PBS liquid is sufficiently mixed by 2~3 times of hemodilution The anticoagulant venous blood of 6mL is slowly superimposed on dropper along tube wall to the 10mL centrifuge tube that 4mL lymphocyte separation medium has been added after even Horizontal centrifugal (400r/min, 20 DEG C) 35min in middle horizontal centrifuge；It is divided into 3 layers after centrifugation in pipe, upper layer is blood plasma and PBS Liquid, lower layer are mainly red blood cell and granulocyte, and middle layer is lymphocyte separation medium, are had in upper, middle layer interface one with single core White cloud and mist layer narrow band based on cell is PBMC, is inserted into cloud and mist layer with capillary syring, draw PBMC be placed in another 50mL from It in heart pipe, is added 5 times and (300r/min, 20 DEG C) 10min is centrifuged with upper volume PBS, abandon supernatant 50mLPBS and cell, centrifugation is resuspended (350r/min, 20 DEG C) 15min abandons supernatant, is added Buffer (PBS+0.5% newborn bovine serum+2mmol/LEDTA, pH7.2) Cell is resuspended in 2mL, takes 15uL cell suspension that the cell (PBMC) counted in 4 block plaids under microscope is added on blood counting chamber Sum.3. CD4+T cell and CD8+T cell isolate and purify: PBMC cell suspension, which is divided equally to two 1.5mLEppendorf, manages, It is centrifuged (300r/min, 20 DEG C) 10min, is discarded supernatant, the every 80uLBuffer of cell is resuspended and contains cell number 10⁷It is a, every 10⁷It is a thin Born of the same parents add 20uLCD4MicroBeads or CD8MicroBeads, mix well, and in 4~8 DEG C of hatching 15min, are washed with 1mLBuffer Cell is washed, (300r/min, 20 DEG C) 10min is centrifuged, 500uLBuffer is discarded supernatant and cell is resuspended, MS splitter is placed on It in the magnetic field of MACS separator, is rinsed with 500uLBuffer, by 500uL cell suspension by splitter, uses 500uLBuffer It rinses splitter repetitive operation 3 times, collects efflux, contain non-CD4+T lymphocyte or non-CD8+T lymphocyte in efflux, Splitter is taken out from separator, with 1000uLBuffer pressure flush splitter, collects efflux, this is thin for CD4+T lymph Born of the same parents or CD8+T lymphocyte (cell viability detection: take 15uL cell suspension and isometric trypan blue molten respectively before and after cell purification Liquid mixing, the not colored shinny person of microscopically observation are living cells, and the coloring person of swelling is dead cell, are calculated living in 200 cells The percentage of cell).

3, amplification in vitro CD4+T cell

The stimulant for thering is document report to grow using the monoclonal antibody of T cell surface C D3 molecule as cell, great Liang Pei After the T cell for supporting AIDS patient's separation, fed back as itself therapeutic cells.But HIV is also with the culture of HIV infection cell It breeds in endogenous multiplication, the feedback of increment T cell also results in the feedback of increment HIV.The present invention is with SV40 and/or hTERT CD4+T cell is immortalized, and using CD3 monoclonal antibody as cell growth stimulant, massive amplification CD4+T cell.

Be by the method for cell growth stimulant of CD3 monoclonal antibody: by anti-CD49d McAb, (CD4+T cell contains simultaneously CD3 molecule) it is coated with to stimulation mononuclearcell (lymphocyte) growth on culture plate, referred to as anti-cd 3 antibodies are coated with method, available Good expanding effect, the lymphocyte that should be expanded in this way have been used for the second stage of clinical treatment of tumour and achieve certain Curative effect.Foreign literature reports [Shimizu etc.] also with the lymphocyte of 5 full-blown AIDS patients of the method culture, training In the cell mass supported and be achieved with 1000 times of amplification for 4 weeks, and expand CD4+/CD8+T can massive amplification (CD4+T cell is more Obviously).Another kind is the dual anti-cross-linking method of AntiCD3 McAb/CD28, i.e., grows AntiCD3 McAb/CD28 dual anti-be crosslinking on pearl as stimulant training It supports HIV infection person's peripheral blood mononuclear cells (lymphocyte), a large amount of CD4+T cell, and the CD4+T expanded can be expanded Cell has the ability of confrontation HIV infection, and virus finds that this may be with CD28 also below detection level later in incubation Second signal is provided, a large amount of Th1 cell factor of selective induction secretion is related with chemotactic factor (CF), is expanded with the method The clinical treatment that CD4+T cell has been used for HIV infection person is fed back, devoid of risk but effect is general.

It is in the method that hTERT immortalizes CD4+T cell: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo- HTERT and carrier pLXSNneo, the hTERT and pLXSNneo separated with Ligation Mix connection through PCR amplification, gel electrophoresis Digestion products construct pLXSNneo-hTERT recon, and it is green to expand, purify simultaneously picking resistant to ammonia benzyl to convert DH5a competent cell Mycin bacterium colony extracts plasmid, imports the T lymphocyte that in vitro passage is in logarithmic growth with lipofection, makes recon and thin The DNA of born of the same parents is integrated, and expands the clone for the positive recombinant that culture is screened through G418, screens cellular morphology, growth curve, dyeing It is body caryogram, the test of nude mice tumorigenesis, transfection Cell Telomerase Activity, hTERT mRNA expression product, immunohistochemical staining, thin Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic and person same or similar with primary cell is as hTERT immortality The CD4+T cell of change.

It is in the method that SV40 immortalizes CD4+T cell: is connected simultaneously with T4DNA ligase through BamHI digestion The SV40LTag DNA of pcDNA3.1 (-) DNA and PCR amplification, agarose gel electrophoresis separation, construct SV40LTag- PcDNA3.1 (-) recombinant plasmid, it is amp-R to expand, purify simultaneously picking to convert DH5a competent escherichia coli cell Bacterium colony extracts plasmid, and the T lymphocyte of in vitro culture is imported with lipofection, integrates the DNA of recon and cell, with The cell containing positive recombinant of G418 screening passes on, expands culture, screens cellular morphology, cell growth curve, chromosome Caryogram, the test of nude mice tumorigenesis transfect the big T genetic test of SV40 in cell DNA, the measurement of mRNA expression product and determined dna sequence As a result meet immortalized cells characteristic and CD4+T cell that person same or similar with primary cell immortalizes as SV40.

1. immortalizing the specific method of CD4+T cell with hTERT

(I) extraction of hTERT: (i) digestion pClneo-hTERT:hTERT be located at the EcoRI of plasmid pClneo-hTERT with Between Sal I site, pLXSNneo vector multiple cloning site (MCS) is containing EcoRI and XhoI restriction enzyme site.Commercially available purchase PCIneo-hTERT plasmid is dissolved in suitable ultra-clean H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds each 0.5ul of restriction enzyme EcoR I and Xho I, and 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator are added 5uL electrophoresis sample loading buffer (can also be by the way that 0.5mol/L EDTA is added) is terminated and is reacted, routinely after PCR method amplification hTERT, Amplified matter is collected in case electrophoresis.(ii) it hTERT electrophoresis: takes electrophoresis grade agarose to be made into 10% agarose with electrophoretic buffer and coagulates Glue pours on the gel casting platform sealed, plugs sample comb, removes envelope band from glue platform after being gelled admittedly, extracts Comb is put into the electrophoresis tank added with enough electrophoretic buffers, and buffer is higher by gel surface about 1mm, with suitable 10 × Sample loading buffer prepares hTERT digestion sample, then sample is added in sample well with pipettor, and do suitable standard simultaneously Reference material connects electrode, and so that hTERT is faced south, Ghandler motion is dynamic, and then electrophoresis divides to enough under the voltage of 1-10V/cm (80V) gel When with a distance from hTERT segment (30min), power supply is closed.(iii) hTERT purifying is with recycling: hTERT is separated from agarose Band: cutting the gel-tape of the segment of hTERT containing target under long wave ultraviolet light source and be fitted into bag filter, adds into bag filter Enter 2ml electrophoretic buffer, be allowed to submerge gel, and empty steam bubble, bag filter level is put into electrophoresis tank (length direction and electrophoresis In parallel), be added appropriate amount of buffer solution by bag filter submerge (about 6-7mm), power on, 150 volts of electricity are washed, observe in the UV lamp to HTERT all removes gel, changes direction of an electric field and continues to be powered 1 minute, buffer is sucked out from bag filter in 1-5ml In Eppendorf pipe, 1.5 times of volume n-butanols are added, mixes extracting and removes EB, most high speed 2 minutes, suck on desk centrifuge Upper layer butanol solution, so repeat it is secondary, be added from the solution of lower layer hTERT isometric phenol chloroform (2) extract 2 times, Supernatant, which is transferred in another Eppendorf pipe, is added 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohols, in 20 DEG C of mistakes Night, 12000g are centrifuged 10 minutes at 4 DEG C, are obtained hTERT precipitating, are abandoned supernatant, abandon dry ethyl alcohol after being added 70% ethanol washing 2 times, add Enter 50 μ l TE dissolution hTERT.In addition, also can be used low melting-point agarose gel method, hTERT filter membrane inserted sheet method etc. by purpose hTERT Segment is separated from gel, is purified.

(II) hTERT composition (0.1-5 μ g), 1 μ of the 9 above-mentioned purifying of μ l the connection of hTERT and pLXSNneo carrier: are taken L10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C incubate for 24 hours, construct pLXSNneo-hTERT recon.

(III) purifying, amplification, the identification of pLXSNneo-hTERT recon: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or a variety of divalent cations etc. handle bacterium, feed them into competence and are converted, and use CaCl₂Fresh or freezing competent escherichia coli cell is prepared, preparation in quantity competent bacteria is usually used in, this law is suitable for big Most coli strains, operating process are summarized as follows: one single colonie of picking in the fresh plate of 16~20h is cultivated from 37 DEG C (such as bacillus coli DH 5 2) or 1ml fresh 16~20h overnight culture, go to the 1L containing 100mlLB culture medium or In 500ml flask, in 37 DEG C of acutely shaking cultures about 2~3h (200~300r/min of rotary shaker), surveyed every 20~30min OD600 value ≈ 0.4 is measured, bacterium is aseptically transferred to one, in ice-cold 50ml polypropylene centrifuge tube, in ice 10~20min of upper placement is centrifuged in 4 DEG C with SorvallGS2 rotary head (or the rotary head to match with its centrifuge tube) with 4000r/min 10min, to recycle cell, culture solution reciprocal is used by pipe inversion 1min so that the trace culture solution of final residual is flow to end with 10ml The 0.1mMCaCl of ice pre-cooling₂Every part of precipitating is resuspended, is put on ice, in 4 DEG C with (or its corresponding turn of SorvallGS3 rotary head Head) with 4000r/min centrifugation 10min pour out culture solution to recycle cell, by pipe be inverted 1min so that final residual trace Culture solution is flow to end, the 0.1M CaCl that every 50ml initial incubation object 2ml ice is pre-chilled₂It is resuspended every part and sinks and determine, at this point it is possible to rapidly Cell is distributed into aliquot, is freezed in liquid nitrogen, -70 DEG C of storages are spare.(ii) with competent E.coli purifying, amplification PLXSNneo-hTERT recon: 200 μ l are taken to be transferred to nothing from every kind of competent cell suspension with cooling sterile pipette tip In the microcentrifugal tube of bacterium, every pipe adds DNA or connection reaction mixture (volume≤10 μ l, DNA≤50ng), gently rotates with mixed Even content, places 30min in ice, and centrifuge tube is put into pre-heating to the rack for test tube in 40 DEG C of circulator bath, places 90s~2min not shake test tube, quickly pipe is transferred in ice bath, make the cooling 1~2min of cell, and every centrifuge tube adds 800 μ Culture medium is warmed to 37 DEG C with water-bath, then pipe is transferred on 37 DEG C of shaking tables by lSOC culture medium, and incubating 45min makes bacterium Recovery, and antibiotic-resistance marker's gene of expression plasmid coding, proper volume (every 90mm plate is up to 200 μ l) has been turned The competent cell of change is transferred on the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, and plate is placed in room temperature It is absorbed to liquid, is inverted plate, cultivated in 37 DEG C, may occur in which bacterium colony after 12~16h.(iii) it screens, expand recon: using Sterile toothpick or disinfection inoculation pin select single bacterium colony and are inoculated in the sterile LB culture medium of 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 culture medium containing LB (containing appropriate antibiotic) In 2L flask, then at 37 DEG C of cultures to saturation state (OD₆₀₀≈ 4, to improve yield, Ying Caiyong surface area is larger and with baffle plate Flask to increase venting quality as far as possible, shake speed should be greater than 400r/min), in 4 DEG C, 6000g is centrifuged 10min, with 4mL GTL Precipitating is resuspended in solution, and is transferred in volume >=20mL high speed centrifugation pipe that (bacterial precipitation can be at -20 DEG C or -70 DEG C Indefinite duration saves), the GTE solution for the lysozyme containing 25mg/mL that 1mL newly matches is added, precipitating is resuspended, in being placed at room temperature for 10min, adds Enter 10mL and newly match NaOH/SDS solution, and mix gently until liquid become uniform, limpid and sticky, placed on ice 10min, be added 7.5mL acetic acid solution, with suction pipe be gently mixed until viscosity decline and formed big precipitating, placed on ice 10min, in 4 DEG C, 20 000g are centrifuged 10min, supernatant are gently poured into another clean centrifuge tube, if there is visible Drift can use several layers of filtered through gauze, the isopropanol of 0.6 times of volume is added, is mixed by inversion, is placed at room temperature for 5~10min, in room Temperature, 1500g are centrifuged 10min, and 70% ethyl alcohol of 2mL is added and gently washs precipitating, then of short duration rapid centrifugation, sucks ethyl alcohol, vacuum Dry (precipitating can be in 4 DEG C of long-term preservations).(iv) identification and amplification of recombinant plasmid: the single colonie on picking plate is inoculated in It in the 3ml LB culture medium of ampicillin containing 100ug/ml, 37 DEG C, cultivates in 250r/min shaking table, collects culture after 14h, 4 DEG C, 10000r/min be centrifuged 5min, extracted in a small amount by kit specification and purify recombinant plasmid；It is bis- with EcoRI and HindIII Digestion recombinant plasmid reaction system: each 0.5ul of restriction enzyme, 10 × buffer 2ul, recombinant plasmid 10ul, Jia Shui are supplied To 20ul, 37 DEG C of digestion 1h.Digestion products carry out 0.8% agarose electrophoresis under 80V voltage conditions, time 30min, gel at As system is taken pictures；Routinely measure the sequence of recombinant plasmid；Recombinant plasmid will contain the matter after digestion, sequencing identification are accurate The microbionation of grain is into LB culture solution, amplification cultivation, carries out large dosage of plasmid by large dosage of plasmid extraction kit specification Extracting and purifying, ultraviolet specrophotometer measure spare after plasmid concentration and purity.

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

(V) CD4+T cell preculture: by above-mentioned cell inoculation in containing 5~10nmol/L insulin, 20% fetal calf serum In RPMI1640 liquid, or be inoculated in containing 20% fetal calf serum, 5~10nmol/L insulin low sugar DMEM cell culture medium in, Generally it is inoculated in culture medium (the 1.6%1M HEPES buffer solution of 3ml Fresh；15% fetal calf serum (FBS)；1% penicillin And streptomysin；PRMI 1640 is supplied to 100%), being placed in 37 DEG C, in volume fraction 5%CO2 incubator, is cultivated 1-2 days, from The heart removes supernatant, spare.

(VI) pLXSNneo-hTERT recon imports T lymphocyte and expands culture: making in 1.5ml microcentrifugal tube Standby following solutions: pLXSNneo-hTERT recon is dissolved in 100 μ l serum-free mediums by pipe A；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, and pipe A and pipe B is mixed, 45min is stood at room temperature, is trained with serum-free Nutrient solution washing above-mentioned T lymphocyte 2 times.1ml serum-free is added in Lipofectamine-pLXSNneo-hTERT mixture Culture solution mixes gently, and is added dropwise in above-mentioned T lymphocyte, and 1ml serum-free medium is added, and (fetal calf serum concentration is 20ml/ L), in CO₂Transfection liquid is sucked out in incubator culture 10h, adds 4ml complete culture solution (fetal calf serum concentration is 20%), continues to cultivate 20h, discards culture solution, and replacement concentration is 400mgL^-1G418 culture solution continue to cultivate, after 8 days select living cells work expand After culture, then G418 concentration is increased to 800mgL^-1, will stablize in the G418 environment of high concentration growth cell continue into Row amplification cultivation.Culture 9 days or so is under the microscope, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If cell increases Slowly or cell density is low or medium pH value is in acidity, and half amount culture solution is sucked out, carries out equivalent oil changing.When total amount reaches 14ml When be transferred in 75ml culture bottle, every 2-3 weeks addition 5-10ml fresh culture.Cell culture to 9-10 weeks (the about the 75th generation), Still in logarithmic growth phase, i.e. cell is accelerated with incubation time in multiplication relation, and dead cell (passes through reading less than 10% The scale of culture vessel judges the increase situation of cell quantity；Identify dead cell and living cells by trypan blue staining.Because Normal living cells, after birth structural integrity can be repelled, and enter trypan blue can not intracellular；And the cell of loss of activity, born of the same parents The permeability of film increases, and can dye blue by trypan blue, can determine whether for cell it is dead.Method be draw weekly it is a certain amount of Suspension culture mixes postposition room temperature 5~10 minutes with Trypan Blue agent, cell sheet is then made, under the microscope 1000 total number of cells are counted, the dead cell of coloring and the percentage of non-staining living cells are calculated).Hereafter with culture algebra Increase and incubation time extension, the increase of cell quantity is slack-off, dead cell is more and more, until cell is not further added by, Even dissolution, reduction, all death.It when total amount reaches 45ml, sets in 50ml centrifuge tube, 1500 turns of centrifugation, 10 minutes, in abandoning After clear, 3ml freezing media (5% dimethyl sub-maple (dimethyl sufoxide), 95%FBS) is added and mixes, it is outstanding at cell (cell concentration is about 10 to supernatant liquid⁵/ml).Cryopreservation tube packing, 1ml/ pipe, set -20 DEG C of 2h, then set -70 DEG C of 2h, then freeze - In 196 DEG C of liquid nitrogen (or immediately under zero setting after 80 degree, 1-2 hours move into liquid nitrogen container in).

(VII) immortalize the identification of CD4+T characteristics of cell biology: (i) observes cellular morphology: visible lymphocyte is obvious Increase, clustering phenomena occur, there is lymphocyte blastogenesis feature.(ii) it observes cell growth curve: taking well-grown transfection Cell suspension is made in cell, through counting, takes 1.4 × 10 respectively⁴Cell inoculation is trained in 30 DMEM culture mediums of low sugar containing 15FBS Support bottle.It takes 2 bottles of cells to be counted daily, calculates mean value, be observed continuously until cell quantity is decreased obviously, after culture 3 days often Liquid is changed every 2 days cells to no count, transfection cell is observed in hepato ZYME-SFM serum free medium using same method In growing state.As a result using incubation time as horizontal axis, cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and is made After curve the cell growth curve, immortalized cell line is in that typical " S " feature or " arched roof " are formed；(iii) in pipe, 1.5 times of volume n-butanols are added, mixes extracting and removes EB, it is molten to suck upper layer n-butanol for most high speed 2 minutes on desk centrifuge Liquid, so repeat it is secondary, from lower layer speech DNA solution in be added isometric phenol chloroform (2) extract 2 times, supernatant is transferred to another 1/10 times of volume 3M NaAc, 2 times of volumes pre-cooling dehydrated alcohols are added in Eppendorf pipe, overnight in 20 DEG C, 12000g, 4 DEG C Lower centrifugation 10 minutes obtains DNA precipitating, abandons supernatant, dry ethyl alcohol is abandoned after being added 70% ethanol washing 2 times, and 50 μ l TE dissolution is added DNA.In addition, low melting-point agarose gel method, DNA filter membrane inserted sheet method etc. also can be used to separate target DNA fragment from gel, is pure It dissolves and.

(II) connection of SV40 large T antigen DNA and pcDNA3.1 genophore: take the above-mentioned DNA composition of 9 μ l (0.1-5 μ g), 10 2 × connection of μ l buffers, 1 μ l 10mmol/L ATP, T4DNA ligase (20~500 cohesive end unit) or large intestine bar Bacterium DNA ligase, the mixing of pcDNA3.1 empty carrier, 15 DEG C incubate for 24 hours, are built into SV40T/pcDNA3.1 recon.

(III) amplification, separation and identification of SV40T/pcDNA3.1 recon: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or a variety of divalent cations etc. handle bacterium, feed them into competence and are converted, and use CaCl₂Fresh or freezing competent escherichia coli cell is prepared, preparation in quantity competent bacteria is usually used in, this law is suitable for big Most coli strains, operating process are summarized as follows: one single colonie of picking in the fresh plate of 16~20h is cultivated from 37 DEG C (such as bacillus coli DH 5 2) or 1ml fresh 16~20h overnight culture, go to the 1L containing 100mlLB culture medium or In 500ml flask, in 37 DEG C of acutely shaking cultures about 2~3h (200~300r/min of rotary shaker), surveyed every 20~30min OD600 value ≈ 0.4 is measured, bacterium is aseptically transferred to one, in ice-cold 50ml polypropylene centrifuge tube, in ice 10~20min of upper placement is centrifuged in 4 DEG C with SorvallGS2 rotary head (or the rotary head to match with its centrifuge tube) with 4000r/min 10min, to recycle cell, culture solution reciprocal is used by pipe inversion 1min so that the trace culture solution of final residual is flow to end with 10ml The 0.1mMCaCl of ice pre-cooling₂Every part of precipitating is resuspended, is put on ice, in 4 DEG C with (or its corresponding turn of SorvallGS3 rotary head Head) with 4000r/min centrifugation 10min pour out culture solution to recycle cell, by pipe be inverted 1min so that final residual trace Culture solution is flow to end, the 0.1M CaCl that every 50ml initial incubation object 2ml ice is pre-chilled₂It is resuspended every part and sinks and determine, at this point it is possible to rapidly Cell is distributed into aliquot, is freezed in liquid nitrogen, -70 DEG C of storages are spare, outstanding from every kind of competent cell with cooling sterile pipette tip Respectively take 200 μ l to be transferred in sterile microcentrifugal tube in liquid, should every Guan Zhongjia DNA or connection reaction mixture (volume≤ 10 μ l, DNA≤50ng), it gently rotates to mix content, 30min is placed in ice, centrifuge tube is put into pre-heating to 40 DEG C Circulator bath in rack for test tube on, place 90s~2min, not shake test tube, quickly pipe is transferred in ice bath, make cell Cooling 1~2min, every centrifuge tube add 800 μ lSOC culture mediums, culture medium are warmed to 37 DEG C with water-bath, is then transferred to pipe On 37 DEG C of shaking tables, incubating 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, by appropriate bulk The competent cell that product (each 90mm plate is up to 200 μ l) has converted is transferred to containing 200mmol/LMgSO4 and corresponding antibiotic SOB culture medium on, plate is placed in room temperature to liquid and is absorbed, plate is inverted, is cultivated in 37 DEG C, may occur in which after 12~16h Bacterium colony.(ii) screening, amplification and extraction of recon: single bacterial examination is selected with sterile toothpick or disinfection inoculation pin and looks into chromosome: By analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY " then illustrate that the cell line does not have Generation vicious transformation (while whether there is abnormal DNA group in available flow cytometry analysis cell line, if not provided, Illustrate that cell line does not occur tumor feature).Chromosome karyotype analysis method is: by the 250ug/ that preheating is added in 5mL culture solution Ml colchicine 100ul mixes 37 DEG C of postposition incubator 4 hours, is centrifuged, removes supernatant, is hypotonic, is fixed, film-making, the aobvious band of G Post analysis karyotype；(iv) Flow cytometry: the cell proportion of synthesis, division in the 19th continuous cell line of detection, such as For its proliferative capacity of fruit obviously than not building the normal cell for being enhancing, explanation is the result of hTERT integration, expression.(vii) DNA sequence Column measurement: routinely sequenator detects, and shows hTERT gene order.(v) hTERT detection in cell DNA is transfected: such as with immune Histochemistry's detection, the visible a large amount of brown particles of the interior dyeing of the nucleus of hTERT transfection, shows that hTERT has been integrated into the cell； (vi) mRNA expression product measures: taking the pcr amplification product of 100 μ l systems, is returned with gel reclaims kit (Takara, Japan) Product is received, 2 μ l DNA solutions is taken to dilute 100 times, surveys concentration, remaining DNA and each 10 μ l of upstream and downstream primer are sequenced.

(VIII) hTERT mediates CD4+T cell bank: screening and continue passage, expansion culture meets forever after above-mentioned identification OEG cell characteristic and the cell same or similar with primary cell, take that growth conditions are good, the difference in logarithmic growth phase The cell of generation is centrifuged (1 200r/min, 6min), and cell is resuspended with 0.5~1ml of frozen stock solution containing dimethyl sulfoxide, Cell density is 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；- 70 DEG C, overnight, enter -196 DEG C of liquid nitrogen It freezes, it is spare to construct the stable immortalization CD4+T cell bank of biological characteristics in this way.

2. immortalizing the specific method of CD4+T cell with SV40

(I) extraction of SV40 large T antigen DNA: (i) SV40DNA digestion: contain large T antigen gene from commercially available purchase SV40 freeze dried powder or SV40 plasmid, are dissolved in suitable H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds restriction enzyme BamH I (1-5U/ugDNA), and 5uL is added in 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator Electrophoresis sample loading buffer (can also be by the way that 0.5mol/L EDTA is added) terminates reaction in case electrophoresis.(ii) it SV40DNA electrophoresis: takes Electrophoresis grade agarose is made into 10% Ago-Gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs sample Product comb removes envelope band from glue platform after being gelled admittedly, extracts comb, be put into the electrophoresis tank added with enough electrophoretic buffers In, buffer is higher by gel surface about 1mm, DNA sample is prepared with suitable 10 × sample loading buffer, then with pipettor by sample Product are added in sample well, and do suitable DNA molecular amount standard control object simultaneously, connect electrode, keep the DNA Ghandler motion that faces south dynamic, Under the voltage of 1-10V/cm gel electrophoresis to be sufficiently separated DNA fragmentation apart from when, close power supply.(iii) divide from agarose From about 2600bp SV40 large T antigen DNA: (using 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 is fitted into bag filter, into bag filter, addition 2ml electrophoretic buffer, makes Submergence gel, and empty steam bubble, bag filter level be put into electrophoresis tank (length direction is parallel with electrophoresis), appropriate buffering is added Bag filter is submerged (about 6-7mm) by liquid, is powered on, and 150 volts of electricity are washed, and observes all remove gel to DNA in the UV lamp, change Power transformation field direction continues to be powered 1 minute, and buffer is sucked out from bag filter and falls that be inoculated in 5mL sterile in 1-5ml Eppendorf LB culture medium or rich medium (such as super broth or TB super broth culture medium) in, after overnight incubation, be then added to In the 2L flask of 500mL culture medium containing LB (containing appropriate antibiotic), then at 37 DEG C of cultures to saturation state (OD₆₀₀≈ 4, to mention High yield, for the larger and flask with baffle plate of Ying Caiyong surface area to increase venting quality as far as possible, shake speed should be greater than 400r/ Min), in 4 DEG C, 6000g is centrifuged 10min, is resuspended and is precipitated with 4mL GTL solution, and is transferred to volume >=20mL high speed In centrifuge tube (bacterial precipitation can be saved in -20 DEG C or -70 DEG C of indefinite duration), the lysozyme containing 25mg/mL that 1mL newly matches is added Precipitating is resuspended in GTE solution, in being placed at room temperature for 10min, 10mL is added and newly matches NaOH/SDS solution, and mixes gently until liquid Body becomes uniform, limpid and sticky, and in placing 10min on ice, 7.5mL acetic acid solution is added, and is gently mixed with suction pipe until viscous Consistency declines and is formed big precipitating, and in placing 10min on ice, in 4 DEG C, 20 000g are centrifuged 10min, and supernatant is gently poured into In the centrifuge tube clean to another, if there is visible drift can use several layers of filtered through gauze, the isopropyl of 0.6 times of volume is added Alcohol is mixed by inversion, and is placed at room temperature for 5~10min, and in room temperature, 1500g is centrifuged 10min, and 70% ethyl alcohol of 2mL is added and gently washs and sinks It forms sediment, then of short duration rapid centrifugation, sucks ethyl alcohol, and is dried in vacuo (precipitating can be in 4 DEG C of long-term preservations).(iii) mirror of recon It is fixed: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competent E.coli, ibid method restriction enzyme Enzyme BamH I carries out digestion, and the identification of 10g/L agarose gel electrophoresis obtains 2 bands of size about 2600bp and 5600bp, the former Meet the size of SV40T segment in GenBank.

(VI) importing and expansion culture of SV40T/pcDNA3.1: following solutions are prepared in 1.5ml microcentrifugal tube: pipe SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free mediums (fetal calf serum concentration is 20ml/L) by A；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, pipe A and pipe B is mixed, the underlying 45min of room temperature.Use free serum culture Liquid washing 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 is added dropwise in above-mentioned T lymphocyte, and 1ml serum-free medium is then added, and (fetal calf serum concentration is 20ml/L), in CO₂Transfection liquid is sucked out in incubator culture 10h, adds 4ml complete culture solution (fetal calf serum concentration is 20%), after Continuous culture 20h, discards culture solution, and replacement concentration is 400mgL^-1G418 culture solution continue to cultivate, select living cells after 8 days Make after expanding culture, then increases G418 concentration to 800mgL^-1, the cell of growth will be stablized in the G418 environment of high concentration Continue amplification cultivation.Culture 9 days or so is under the microscope, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If thin Born of the same parents increase slowly or cell density is low or medium pH value is in acidity, are sucked out half and measure culture solution, carry out equivalent oil changing.When total amount reaches It is transferred in 75ml culture bottle when to 14ml, every 2-3 weeks addition 5-l0ml fresh culture.Cell culture about 6-8 weeks the (the about the 55th Generation), still in logarithmic growth, i.e. incubation time and cell is accelerated in multiplication relation, and dead cell (passes through reading less than 10% The scale of culture vessel is taken to judge the increase situation of cell quantity；Identify dead cell and living cells by trypan blue staining.Cause For normal living cells, after birth structural integrity can be repelled, and enter trypan blue can not intracellular；And the cell of loss of activity, The permeability of after birth increases, and can dye blue by trypan blue, can determine whether for cell it is dead.Method be draw weekly it is a certain amount of Suspension culture, mix postposition room temperature 5~10 minutes with Trypan Blue agent, cell sheet be then made, in microscope 1000 total number of cells of lower counting, calculate the dead cell of coloring and the percentage of non-staining living cells).Hereafter with culture generation The extension of several increase and incubation time, the increase of cell quantity is slack-off, dead cell is more and more, until cell no longer increases Add, or even dissolution, reduction, all death.It when total amount reaches 45ml, sets in 50ml centrifuge tube, 1500 turns of centrifugation, 10 minutes, Supernatant is abandoned, 3ml freezing media (5% dimethyl sub-maple (dimethyl sufoxide), 95%FBS) is added and mixes, at cell (cell concentration is about 10 to suspension⁵/ml).Cryopreservation tube packing, 1ml/ pipe, sets -20 DEG C of 2h, then set -70 DEG C of 2h, then freezes In -196 DEG C of liquid nitrogen (or immediately under zero setting after 80 degree, 1-2 hours move into liquid nitrogen container in).

(VII) immortalize the identification of CD4+T characteristics of cell biology: (i) observes cellular morphology: visible lymphocyte is obvious Increase, clustering phenomena occur, there is lymphocyte blastogenesis feature.(ii) it observes cell growth curve: taking well-grown transfection Cell suspension is made in cell, through counting, takes 1.4 × 10 respectively⁴Cell inoculation is trained in 30 DMEM culture mediums of low sugar containing 15FBS Support bottle.It takes 2 bottles of cells to be counted daily, calculates mean value, be observed continuously until cell quantity is decreased obviously, after culture 3 days often Liquid is changed every 2 days cells to no count, transfection cell is observed in hepato ZYME-SFM serum free medium using same method In growing state.As a result using incubation time as horizontal axis, cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and is made After curve the cell growth curve, immortalized cell line is in that typical " S " feature or " arched roof " are formed；(iii) it checks Chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY " then illustrate the cell There is no vicious transformations (while abnormal DNA group whether occur in available flow cytometry analysis cell line, not have such as system Have, illustrate that cell line does not occur tumor feature).Chromosome karyotype analysis method is: preheating by being added in 5mL culture solution 250ug/ml colchicine 100ul, mix 37 DEG C of postposition incubator 4 hours, be centrifuged, remove supernatant, is hypotonic, is fixed, film-making, The aobvious band post analysis karyotype of G；(iv) Flow cytometry: the cell ratio of synthesis, division in the 19th continuous cell line of detection Example, if its proliferative capacity is not obviously than building the normal cell for being enhancing, explanation is the result of the integration of SV40 large T antigen, expression. (viii) determined dna sequence: routinely sequenator detects, and shows SV40 large T antigen DNA sequence dna.(v) it transfects in cell DNA The big T genetic test of SV40: such as with Immunohistochemical detection, the nucleus of SV40 transfection is interior to dye visible a large amount of brown particles, Show that SV40T antigen has been integrated into the cell；Expression of the RT-PCR method detection T antigen in cell can also be used, wherein T antigen Primer: upstream primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', downstream primer (S4496) 5 '-GAA ATG CCA TCT AGT GAT-3'；Amplified production length is 268bp, and 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⁺] 200 μm of ol/L of 2mmol/L, dNTPs, 0.4 μm of ol/L, Taq1U of primer concentration, 5 μ l of template；Experimental group By template of the cDNA of the 19th generation cell (synthesis of the first chain of cDNA, product-are carried out referring to commercially available cDNA the first chain synthetic agent box 20 DEG C of preservations)；Negative control sets two, does template respectively with the cDNA of sterile water, primary cell, positive control is with SV40DNA For template (extract SV40DNA referring to SDS- proteinase-K pathway because SV40 virus without coating, does not use SDS rupture of membranes, take 5 μ l into The detection of 1.5% agarose gel electrophoresis of row, remaining -20 DEG C save backup)；(vi) mRNA expression product measures: T antigen mRNA The sequencing of RT-PCR product: taking the amplified production of 100 μ l systems, with gel reclaims kit (Takara, Japan) recovery product, takes 2 μ l DNA solutions dilute 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer are sequenced.

(VIII) SV40LT gene mediated CD4+T cell bank: screening and continues passage, expands culture accords with after above-mentioned identification Close immortalized cells characteristic and the cell same or similar with primary cell, take growth conditions it is good, in logarithmic growth phase The cell of different generations is centrifuged (1200r/min, 6min), is resuspended with 0.5~1ml of frozen stock solution containing dimethyl sulfoxide thin Born of the same parents, cell density are 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；- 70 DEG C, overnight, enter -196 DEG C of liquid Nitrogen freezes, and it is spare to construct the stable CD4+T cell bank of biological characteristics in this way.

(4) with the preparation of CD4+T cell identity function particle: can be by CD4 molecule, the CD4 molecule of genetic recombination and similar The molecule of function is coupled by conventional chemical, is crosslinked, for being made and being coated with CD4 molecule is fixed on carrier in affine absorption etc. Grain, or directly take intimate particle substitution CD4+ cell application.It is thin that CD4+T cell of the invention represents other CD4+ Born of the same parents, including prepared with other methods and immortalize CD4+T cell.

Two, the preparation of AIDS plasma purification device

1, the filling of cleanser

By hybridoma macrophage prepared by the present invention and CD4+T cell, cleaned with sterile saline, 1000r/min Centrifugation is centrifuged 5min again after cleaning with 1000r/min, by the ratio between the strain of hybridoma macrophage and CD4+T cell strain for 1: 0.5~ 3 ratio takes sedimentation cell, is packed into 200ml hydrostatic column made of acrylate etc high molecular material, is fills up to 4/ 5, cylindrical column is made, cell column is sealed it is spare, separately taken carrier function after 100 DEG C dissolve heat preservation 39~42 DEG C 0.7%, 0.8%, 0.9%, 1.0%, 1.1% agarose C1-4B physiological saline, successively taken by high concentration to low concentration Isometric agarose is added in cell column, it is desirable that the agarose being first added be cooled to 37 DEG C become after semisolid just then plus Next time, the layer distributed but daf molecule for making agarose concentration of the cell column from import to outlet formation from low to high account for 4/5, Wherein hybridoma macrophage and CD4+T cell, which are fixed in gel, plays a part of to adsorb HIV.Agar gel is formed by Filter opening is reduced with increasing for agarose concentration, and clarifier entrance agarose concentration is low, and filter opening is just big, is conducive to blood plasma filling The association reaction of stream and cell and HIV；And exit concentration is high, filter opening is just small, is easy to detention HIV or Large molecular conjugates.Only Change device and is combined with a variety of special and non-specific HIV removing compositions, in case extraordinary strain is because of the futile treatment caused by immunity difference.

2, the specification of clarifier

The cell column of above-mentioned preparation is clarifier, is bottom diameter is small, top diameter is big cylinder or rectangular, infundibulate, volume For 200~300ml, inlet and outlet are equipped with cell screen clothes, and entrance top diameter sieve mesh number is 800 mesh；Exit bottom diameter sieve mesh Number be 2.0~5.0 mesh (2.5~5.0 mesh are equivalent to 0.1~0.2 micron or 100~200 nanometers), specifically can be made into 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh 7 kinds of different sizes, to stop 120 nanometers inhibition of HIV or Bigger bacterium；The cell strainer that mesh number is 100 mesh (being equivalent to 4 microns) is arranged in liquid outlet, may filter out to stop Cell；It is equipped with buffer area between liquid entrance and mesh screen, is conducive to the stability of system circulation.

3, the material of clarifier

Blood purification selects the high molecular material of acrylate etc, it is desirable that good biocompatibility, hardly activation are mended Body, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.Can by covalently, The methods of grafting, polymerization improve the structure of material, the inhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxidative stress Influence, to improve biocompatibility, reduce complication generation.Add hydrophilic gel in clarifier inner surface, by 2 methyl-props Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate film, by controlling wet-spinning procedure, is produced CA/PMB30, CA/PMB80 and CA/PMB30-80, blood and cell compatibility with higher.There is anticoagulation by certain Substance be solidificated on the material of carrier or clarifier inner surface, can inhibit blood clotting, improve biocompatibility, can also reduce Heparin dosage, and be possible to realize no-rod tractor.Such as heparin is aggregated on polyacrylonitrile-polyethyleneimine film, effect may Can be more preferable, and the allergic reaction during purification can be reduced, the polyacrylonitrile surface for solidifying chitosan and heparin covalent object is also shown Good blood compatibility, and can inhibit the activity of pseudomonas aeruginosa, reduce cell-cytotoxic reaction.On cellulose acetate film Covalent immobilisation linoleic acid film, or the linoleic acid for being covalently bound to polyacrylic acid is grafted onto polysulfones film surface, can have more preferable Histocompatbility and anticoagulant effect.

Three, the preparation of plasma separator

1, it preparation principle: is prepared according to the molecular size of haemocyte and blood plasma components.Such as visible component (blood in blood of human body Cell) size are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, neutrality About 12 μm of granulocyte, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, and 6-8 μm of small lymphocyte, Approximate with red blood cell, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, 1~4 micron of diameter to 7~8 microns not Platelet mean diameter Deng, people is 2-4 micron, 0.5~1.5 micron of thickness.

2 materials: poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. can be selected, it is desirable that good biocompatibility hardly activates Complement, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.It can be by altogether The methods of valence, grafting, polymerization improve the structure of material, the microinhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxygen Change stress influence, the generation to improve sieving adequacy and biocompatibility, reduce complication.

3, type and spec: for the shape of separator, filter core preparation can be made with materials such as acetate fiber or absorbent cotton The shapes such as flat structure are prepared into as filter core at column construction, with materials such as poly-vinegar non-woven fabrics；By haemocyte and blood to be separated The molecular size of slurry composition determines aperture.It is plasma separator according to the present invention property stabilization, good biocompatibility, penetrating Property high high molecular polymer hollow fibre type filter is made, hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm, Aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm.The hole only permit blood plasma filtration, but can stop all cells at Point.

Four, the component of AIDS plasma purification device

1, key member: (1) plasma separator: for separating mononuclear blood cell and blood plasma；(2) it plasma purification device: is used for HIV in adsorbed plasma.

2, additional member: including blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The parts such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring form.(1) blood pump (Blood Pump): for pushing blood Circulation is to maintain going on smoothly for blood purification treatment, and usual blood pump part often has rotary test speed function, to monitor patient Blood circumstance, therefore blood pump runner and the setting of groove spacing are accurate, and need often adjustment, according to the feelings of bloody path pump line Spacing is generally set as 3.2~3.3mm by condition, can not be too loose, and it is inaccurate otherwise to will cause blood flow detection；Also can not be too tight, otherwise It will cause pipe breakage.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump clinically applied, and uses It to continue the injecting heparin in sieving pipeline (patient blood), contacts, is easy with air since the blood of patient recycles in vitro Blood coagulation phenomenon occurs, anticoagulative can be occurred using heparin pump.(3) sound pulse pressure monitor: arterial blood pressure monitoring mainly to The stopping state of dynamic monitoring blood separator micropore, in addition to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When When blood flow deficiency, angiosthenia will be reduced；When having blood coagulation, thrombosis, especially separator blockage of the micro orifice, angiosthenia will It increases；Vein pressure monitoring is used to monitor the pressure of pipeline blood reflux, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow When insufficient and venous return syringe needle falls off, vein pressure will decline, if bloody path return pipe distortion blocking or reflux syringe needle When blocking, vein pressure will be increased.(4) air monitering (Air Detector): the air gas for monitoring blood pathway Bubble, the general principle for using ultrasonic listening, in order to avoid air embolism occurs for patient and is arranged.When having monitored air bubble When, detection system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent dangerous generation.

In short, Import computer regulates and controls and is made the people of operation on the basis of key member of the present invention and additional member Property, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily judgement is warned Report the blood purifying therapeutical instrument of the micro computers such as reason and ring off signal processing.

Five, the connecting path and application method of AIDS plasma purification device

1, it installs: such as Fig. 1, with sterile working connecting components, including plasma separator, clarifier and each circulation line.

2, it is vented: with sterile saline filling liquid separator, clarifier and each circulation line, excluding separator, clarifier And its gas, bubble in circulation line, it goes through, confirmation after gas, bubble without using.

3, lead to liquid: arterial blood line pipe (1) being connected to the arteries of AIDS patient, in operation the row of going through again Whether gas is complete, and whether liquid stream is unobstructed, and flow liquid in pipe is avoided to pollute.

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

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (5) are connected into venous blood Then pipe opens blood pump (2), blood flow is 100~150ml/min, and such as Fig. 1, arterial blood is through arterial blood line pipe (1) entrance Plasma separator (4), the blood plasma separated reach clarifier (8) through circulation line (7) under the action of blood plasma pump (6), to Full of blood plasma, about 10 minutes, begin releasing blood plasma, flowed out through circulation line (10), it is synchronous that blood plasma is perfused to clarifier (9), When blood plasma in clarifier (8) has nearly flowed, perfusion blood plasma is started again at, clarifier (9) begins releasing blood plasma at this time, and two simultaneously Clarifier (8), the clarifier 9 of connection) alternately, purified blood plasma divides through circulation line (10) and plasma separator (4) From haemocyte converge after through venous line (5) feed back.Such as Fig. 2, when blood to be separated enters the inner cavity of plasma separator (1) (2) when, the effect through valve (8) can enter the exocoel (6) of separator, so by the small molecule blood plasma components (5) of micropore (3) It flows out, and cannot be flowed out through valve (8) by the haemocyte (4) of micropore (3) by plasma outlet port (7).Such as Fig. 3, when containing HIV (1) when blood plasma enters clarifier, HIV (1) therein be fixed on respectively macrophage strain (2) in agar gel (6), CD4+T cell (4) is combined into macrophage phagocytosis object (3), CD4+T cell conjugates (5), and the HIV after being combined is no longer down Mobile, the HIV for 100~120nm not in addition being combined is again by smaller about 1% concentration of 85nm in aperture due to concentration is higher Bottom agar gel molecular Screen Out stay at (7).HIV is so removed, until the plasma circulation amount being previously set is (usually 9L), treatment just end, entire therapeutic process is controlled by computer, and can detect working condition at any time, it is easy to use, oneself Dynamicization and safety.

Six, the verifying of AIDS plasma purification device effect

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

In order to verify the effect of HIV is removed in the absorption of CD4+T cell strain, the present invention devises easy test method: taking and goes out 2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5, draw respectively be centrifuged (1000r/min, 5min) precipitating CD4+T cell extremely 200mm scale is then drawn the heat preservation after 100 DEG C dissolve and is reached about in 39~42 DEG C of 0.9% spare agarose C1-4B 10mm long scale, after setting blood sedimentation stand cooling, agarose becomes semisolid, and blood sedimentation tube inner cell can be prevented to flow out but not prevented small The water of molecule and the substance of chemical analysis etc pass through.The AIDS that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample database save 5 blood plasma of patient, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and 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 layer simultaneously after outflow, collects efflux, blood plasma after referred to as AIDS filter out of blood sedimentation tube.Before taking AIDS to filter Blood plasma after blood plasma and filter, according to HIV-1p24 antigen detection kit, (the limited public affairs of biotechnology are inspired in enzyme-linked immunization, Shanghai Department) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is lower than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity as control, minimum detection limit 450nm measures absorbance (OD) in 0.5pg/ml~80pg/ml, 15min, and blank control calibration object absorbance value is not higher than 0.050,0pg absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbance, is recognized as absorbance > 0.12 For be it is positive, testing result (table 1) illustrate, and after the simple purification device of AIDS blood plasma filtration cell containing CD4+T, part HIV is It is adsorbed by CD4+T cell, after the 1st filtration, HIV total body clearance is 22.84%, and after the 2nd filtration, total body clearance is 35.31%, after the 3rd filtration, total body clearance 41.9%.Illustrate the increase with filtration number, HIV can be by constantly clear It removes, to reach treatment AIDS purpose.

1 AIDS blood plasma of table filters the simple purification device front and back p24 testing result (pg/ml) of the cell containing CD4+T

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

In order to verify the effect of HIV is removed in macrophage strain, the present invention devises easy test method: taking sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draw respectively be centrifuged (1000r/min, 5min) precipitating macrophage hybridoma it is thin Born of the same parents' strain is then drawn the heat preservation after 100 DEG C dissolve and is reached in 39~42 DEG C of 0.9% spare agarose C1-4B to 200mm scale To about 10mm long scale, after setting blood sedimentation tube cooling, agarose becomes semisolid, and blood sedimentation tube inner cell can be prevented to flow out but not hinder Only the water of small molecule and the substance of chemical analysis etc pass through.The Chinese mugwort that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample database save 5 blood plasma of sick (AIDS) patient are grown, respectively about 10mL, blood plasma injects blood sedimentation tube (simple purification in batches before respectively taking 9mLAIDS to filter Device) upper end blank pipe, wait flow through the hybridoma macrophage strain layer of blood sedimentation tube lower layer and out of blood sedimentation tube after outflow, collection outflow Blood plasma after the filter of liquid, referred to as AIDS.Blood plasma and blood plasma after filter before taking AIDS to filter, with human macrophage migration inhibitory factor (MIF) ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) pairing detection, by specification operation, detection range be 0~ 800pg/ml, susceptibility 1.0pg/ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive Stronger, to be colourless or extremely shallow, the depth of the be in color of foundation is indicated negative reaction with "+", "-" number.OD value can also be surveyed: On ELISA detector, at 450nm (if developing the color with ABTS, 410nm), each hole OD value is surveyed after returning to zero with blank control wells, if It is as positive greater than 2.1 times of defined negative control OD value.As a result such as table 1, MIF testing result is yin in blood plasma before filtering Property (or because of content lower than detection sensitivity, blood plasma long-term preservation cause degradation etc.), and testing result is the positive in blood plasma after filtering. Illustrate that macrophage hybridoma cell strain produces MIF cell factor in this process.MIF is collection cell factor, growth factor, swashs Element and the multi-effect protein molecular of enzyme characteristic play central as inherent immunity and the regulatory factor of inflammatory reaction Effect plays panimmunity function in various infection and active chronic inflammation disease.Making the simple purification of AIDS blood plasma filtration Before and after device while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, is detected and is tried according to HIV-1p24 antigen Agent box (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ The p24 antigen of ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are lower than as control, minimum detection limit 5pg/ml, 0~400pg/ml of measurement range, 450nm measures absorbance in the range of linearity 0.5pg/ml~80pg/ml, 15min (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ml absorbance not higher than 0.050,0pg absorbance value It is considered as the positive as absorbance > 0.12 not less than 1.000, as a result (table 2) illustrates the simple purification dress of AIDS blood plasma filtration It postpones, part HIV is swallowed by macrophage hybridoma cell strain to be adsorbed, and the blood plasma HIV after filtration is significantly reduced, through the 1st time After filtration, HIV clearance rate is 20.55%, and after the 2nd filtration, HIV clearance rate is 42.83%, p < 0.01, is had apparent Effect illustrates constantly be removed with the increase HIV of filtration number, to reach treatment AIDS purpose.

1 AIDS blood plasma of table filter MIF testing result before and after the simple purification device of the macrophage containing hybridoma (it is quantitative: pg/ml)

2 AIDS blood plasma of table filters the simple purification device front and back p24 testing result (p24:pg/ of the macrophage containing hybridoma ml)

In short, above-mentioned simple experiment shows that the HIV to dissociate in blood plasma can be by blood purification agent (macrophage of the invention Strain, CD4+T cell strain, agar gel micropore) absorption removing, it is significant clear to show that AIDS plasma purification device of the invention has Except the therapeutic efficiency of blood plasma inhibition of HIV.

Claims

1. a kind of AIDS plasma purification device for medical domain, which is characterized in that by made hybridoma macrophage strain and CD4+T cell strain is formulated in agar gel, and the clarifier of the setting of exit made of high-biocompatibility material filter screen is perfused, The layer distributed but daf molecule for making the agarose concentration of entrance from low to high to exit formation account for the 4/ of overall perfusion object 5, the clarifier constitutes the main part of purging in vitro device with the separator of energy separated plasma and haemocyte, for removing in blood plasma HIV.

2. AIDS plasma purification device according to claim 1, which is characterized in that the volume of the clarifier be 200~ 300ml, entrance are equipped with top diameter cell screen clothes, and top diameter cell screen clothes mesh number is 800 mesh, between entrance and top diameter cell screen clothes Stablize the buffer area of circulation equipped with promotion system, exit is equipped with filter screen, and the filter screen includes bottom diameter cell screen clothes and thin Born of the same parents' strainer, bottom diameter cell screen clothes mesh number are 2.0~5.0 mesh, and cell strainer mesh number is 100 mesh, exit and bottom diameter cell screen clothes Between also be equipped with promotion system stablize circulation buffer area.

3. AIDS plasma purification device according to claim 2, which is characterized in that the exit bottom diameter cell sieve mesh Numeral system at 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh 7 kinds of different sizes.

4. AIDS plasma purification device according to claim 1, which is characterized in that the agarose concentration from low to high Layer distributed refer to the agarose concentration from the entrance of clarifier to exit successively with 0.7%, 0.8%, 0.9%, 1.0%, 1.1% point 5 layers.

5. AIDS plasma purification device according to claim 1, which is characterized in that the daf molecule refers to huge by hybridoma The ratio that the ratio between phagocyte strain and CD4+T cell strain are 1: 3 is prepared.

6. according to claim 1, AIDS plasma purification device described in 5, which is characterized in that the CD4+T cell strain is with SV40 And/or hTERT is prepared as rotaring redyeing gene, using CD3 monoclonal antibody and/or CD28 double antibody as cell growth stimulant.

7. AIDS plasma purification device according to claim 1, which is characterized in that the separator hollow-fiber film is straight Diameter is 270~370 μm, and film thickness is 50 μm, and aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm, can be stopped all Cell filtration.

8. a kind of preparation method of the AIDS plasma purification device cleanser for medical domain, which is characterized in that by hybridoma Macrophage strain and CD4+T cell strain, are cleaned with sterile saline, 1000r/min centrifugation, again with 1000r/min after cleaning It is centrifuged 5min, takes cell precipitation to be packed into the ratio that the ratio between the strain of hybridoma macrophage and CD4+T cell strain are 1: 0.5~3 high In 200ml hydrostatic column made of biocompatible materials, cell column is made, daf molecule is made to account for 4/5, respectively with 100 DEG C 0.7%, 0.8%, 0.9%, 1.0%, the 1.1% agarose C1-4B physiological saline at 56 DEG C is kept the temperature after dissolving, by high concentration To low concentration, successively impartial sampling is added in cell column, it is desirable that the agarose being first added is cooled to after semisolid just then plus next It is secondary, make that cell column forms the layer distributed of agarose concentration from low to high to outlet from import and daf molecule accounts for overall perfusion The 4/5 of object, wherein hybridoma macrophage and CD4+T cell, which are fixed in gel, plays a part of to adsorb HIV.