CN106267424B

CN106267424B - AIDS immune cell therapy instrument

Info

Publication number: CN106267424B
Application number: CN201610540906.1A
Authority: CN
Inventors: 翁炳焕; 李兰娟; 郑杰; 潘小平; 虞晓鹏
Original assignee: 翁炳焕
Current assignee: Yuncheng Tongchuang Medical Laboratory Co., Ltd.
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2019-01-22
Anticipated expiration: 2036-07-01
Also published as: CN106267424A

Abstract

A kind of AIDS immune cell therapy instrument for medical domain, it is characterized in that preparation energy separated plasma, the blood and plasma separator of mononuclear blood cell and the multinucleate giant cell formed because living away from home HIV, it can be in conjunction with the CD4+T cell strain of HIV with the preparation of external source Gene transfer techniques, clarifier is rolled into high-biocompatibility material after amplification, the agarose of gradient concentration is followed by added, fixed CD4+T cell strain therein plays a part of to adsorb HIV, the critical component of extracorporeal circulation apparatus is collectively formed with separator, when blood stream is through blood separator, multinucleate giant cell containing HIV is filtered out, and then when flowing through clarifier by the blood plasma that plasma separator separates, HIV therein is cleaned agent absorption and removes, the purified blood plasma mononuclear blood cell separated with plasma separator is fed back after converging, from And achieve the purpose that the toxin expelling purification treatment for removing the inside and outside HIV of haemocyte.

Description

AIDS immune cell therapy instrument

Technical field

The present invention relates to the preparations and application of AIDS immune cell therapy instrument in medical domain, are mainly used for AIDS sufferer The removing of the inside and outside AIDS virus of person's haemocyte, to achieve the purpose that treatment.

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 the clarifier for the treatment of AIDS after mass propgation, with CD4+T cell absorption HIV and generates cell The factor treats AIDS patient.

Ag-Ab precipitation reaction was initially applied to research Liesegang's phenomenon in 1905 in gel, applied within 1932 In identification bacterium bacterial strain, nineteen forty-six Oudin carries out immunodiffusion in test tube, for analyzing antigen mixture, 1948 Elek and Ouchterlony establishes the two-way double-diffusion process of agar respectively, for identifying simultaneously, more two or more antigens or anti- Body.The media gel agar or agarose of Ag-Ab precipitation reaction in gel are a kind of polysaccharide body containing sulfate, high temperature When can be dissolved in water, gel is congealed into after cold, inside forms a kind of porous reticular structure, and aperture is very big, allow macromolecular Substance (molecular weight is up to million or more) passes freely through.The size in aperture further depends on agar concentration, and agar concentration is big, aperture phase To smaller, agar concentration is small, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, since agar or agarose have Good chemical stability, water content is big after gel, and transparency is good, and convenient sources are easy to handle, therefore is a kind of diffusion well Medium.The molecular weight of antigen and antibody in gel from high concentration region to low concentration region generally all 200,000 hereinafter, spread When suffered resistance very little, be substantially in free diffusing form.When antigen and corresponding antibodies meet in gel after spreading, shape Maximum compound is formed if the two is appropriate in place's ratio of meeting at antigen antibody complex.Due to the molecular weight of compound Increase, particle increase, thus do not continue to spread and generate to precipitate, and show threadiness or band-like, this precipitating is formed one A " specific barrier ", all antigen or antibody molecule same in immunology cannot pass through, and those of property difference Molecule can continue to spread by this barrier, until forming the compound of themselves.Such reaction is known as agar Gel or Immune proliferation are at present with the routine experiment checkup item of known antibodies detection unknown quantity corresponding antigens, and " China Pharmacopeia " standard method of the regulation for the detection of influenza virus vaccine hemagglutinin content in 2010 editions.Usually by a certain amount of goat-anti People's Ig antiserum ingredient is mixed in agar gel, is made containing the specificity sero-fast agar plate of goat-anti people Ig, after to be solidified Punching, and human serum to be checked (IgG, IgA, IgM etc.) is added in corresponding aperture, expand serum to be checked around in agar plate It dissipates, properly locates to combine in antigen and antibody concentration ratio, form macroscopic white precipitate ring and no longer spread.Thus As it can be seen that macromolecular solute therein is just existed by the gel pore detention that molecular sieve acts on when a kind of solution passes through semi-solid gel In gel, antibody that antigen especially therein can be fixed in advance in gel in conjunction with and be attracted in gel.

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 immune cell therapy instrument；It is immune thin that another object is to provide for AIDS The preparation of born of the same parents' therapeutic equipment and application method.

The object of the present invention is achieved like this: it is thin that preparation can filter out the large volume containing HIV that many cells are combined into The blood separator of born of the same parents and the plasma separator that mononuclear blood cell and blood plasma can be separated, with the method building of foreign gene transfection CD4+T cell strain and/or the amplification using the peculiar molecular antibody of CD4+T cell surface as cell growth stimulant, take CD4+T cell Strain with high-biocompatibility material package and prepare can prevent cell and its fragment filter out and can for cell strain absorption HIV field be provided Blood purification, taken keeping the temperature after 100 DEG C dissolve in 39~41 DEG C of 0.7~1.1% gradient concentration for carrier function Agarose, sequentially add blood purification from high to low by concentration, be cooled to 37 DEG C become semi-solid gel after followed by Add the layer distributed for making the gel in clarifier form agarose concentration from low to high from top to bottom next time, is conducive to blood The effect of perfusion and molecular sieve and immune clearance is starched, wherein CD4+T cell strain is fixed in Ago-Gel, plays absorption HIV Effect, prepared AIDS blood purification and then with blood, plasma separator group merge additional computer regulatory process And AIDS immune cell therapy instrument is prepared, the blood separator that the blood in extracorporal circulatory system is treated in instrument filters out large volume Multicore haemocyte, and then blood plasma and mononuclear blood cell are divided by plasma separator, the purified device of blood plasma filter out after HIV with single blood It is fed back after cell confluency.

Technological core of the invention is made of blood, plasma separator and plasma purification device, wherein the hole of blood separator Diameter is 150~250 μm, 50~150 μm, 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm, 1~2 μm, can be filtered out in blood The plastidogenetic multinucleate giant cell of HIV infection or many cells condensate, the single blood that plasma separator can separate medium volume are thin Born of the same parents and blood plasma, the cleanser in plasma purification device is made of agar gel medium and CD4+T cell strain, because of CD4+T cell surface CD4 molecule be HIV receptor and become HIV permissive cell, HIV can be adsorbed when meeting with HIV, the HIV being adsorbed is with CD4 + T cell is fixed in agar gel, and agar gel is formed by filter opening and reduces with increasing for agarose concentration, only Change device 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 exit Concentration is high, and filter opening is just small, is easy to detention HIV or Large molecular conjugates, it is clear that clarifier is combined with a variety of special and non-specific HIV Except composition, in case extraordinary strain is because of the futile treatment caused by immunity difference, so following in vitro in AIDS toxin expelling purification treatment In ring, the large volume cell containing a large amount of HIV is filtered out by blood separator first, and the HIV to dissociate in blood plasma is by plasma purification Device is removed, and the mononuclear blood cell of purified blood plasma and medium volume is fed back after converging, thus remove be incorporated in cell surface with/ Or the intracellular HIV and HIV for being free on blood plasma.The present invention with external machinery filter out HIV method substitution for a long time without Method effectively kills the conventional anti-reverse transcription drug treatment in vivo of HIV, realizes manually the controlling from internal mechanical removal by HIV Treat new method.

Specific embodiment

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

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

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

Fig. 4 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, the other end through heparin and blood pump (2) with contain There is the blood separator (3) of waste liquid outlet (5) to be connected, blood separator (3) exports (4), blood pump (6), circulation pipe through blood Road (7) is connected with plasma separator (8), and the plasma outlet port of plasma separator (8) is through blood plasma pump (9) and blood vessel (10) and two Clarifier (11) in parallel is connected with clarifier (12), the export pipeline (13) of two clarifiers and the blood of plasma separator (8) Cell outlet pipeline (14) converge after through venous line (15) converge body circulation.

Fig. 2 indicates the internal structure of the blood separator (3) in Fig. 1.In Fig. 2, the tube wall of the inner cavity (2) of separator (1) On have many micropores (3), multinucleate giant cell (4) cannot filter micropore (3) and be delayed at inner cavity (2), so as to be removed, energy By in micropore (3), the mononuclear blood cell (5) of small size enter exocoel (6), (7) outflow is then exported, and then through Fig. 1 Shown in plasma separator (8) separation blood cells and blood plasma.

Fig. 3 indicates the internal structure of the plasma separator (8) in Fig. 1.In Fig. 3,1 is plasma separator, and 2 be blood plasma separation Device inner cavity, 3 be the micropore on the tube wall of plasma separator inner cavity, 4 be cannot be by the mononuclear blood cell of micropore (3), 5 be that can pass through The blood plasma chemical analysis of micropore (3), 6 be plasma separator exocoel, and 7 be blood plasma outflux, and 8 be to have the blood of switchable valve thin Born of the same parents outlet.

In Fig. 4,1 is clarifier, and 2 be the CD4+T cell strain being fixed in agar gel, and 3 be to enter fine jade together with blood plasma The HIV of rouge gel, the conjugate no longer moved down after the 4 CD4+T cell strain absorption being fixed in agar gel for HIV, 5 are The cell factor that CD4+T cell strain generates.

Below with reference to Fig. 1, Fig. 2, Fig. 3 and Fig. 4, to the embodiment of AIDS immune cell therapy instrument proposed by the present invention It is explained in detail.

One, the preparation of CD4+T cell strain

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 of the U.S.).

2, the separation (density-gradient centrifugation method) of mononuclearcell (PBMC): it is derived from the fresh blood or new of Blood Center storage The Cord blood that raw youngster discards, take wherein 20mL blood sample (500IU/mL2mL heparin sodium is anticoagulant) it is anticoagulant, PBS liquid by hemodilution 2~ 3 times, 6mL anticoagulation is slowly superimposed on to the 10mL that 4mL lymphocyte separation medium has been added with dropper along tube wall after mixing well Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in centrifuge tube；It is divided into 3 layers after centrifugation in pipe, upper layer is blood plasma With PBS liquid, lower layer is mainly red blood cell and granulocyte, and middle layer is lymphocyte separation medium, is had in upper, middle layer interface one with list White cloud and mist layer narrow band based on a nucleus is PBMC, is inserted into cloud and mist layer with capillary syring, draws PBMC and is placed in another -50mL It in centrifuge tube, is added 5 times and (300r/min, 20 DEG C) 10min is centrifuged with upper volume PBS, abandon supernatant 50mLPBS and cell is resuspended, from The heart (350r/min, 20 DEG C) 15min, abandoning supernatant, addition Buffer (PBS+0.5% newborn bovine serum+2mmol/LEDTA, PH7.2) cell is resuspended in 2mL, takes 15uL cell suspension that the cell counted in 4 block plaids under microscope is added on blood counting chamber (PBMC) total.3. CD4+T cell and CD8+T cell isolate and purify: PBMC cell suspension is divided equally to two 1.5mLEppendorf pipe, is centrifuged (300r/min, 20 DEG C) 10min, discards supernatant, and the every 80uLBuffer of cell is resuspended and contains cell Number 10⁷It is a, every 10⁷A cell adds 20uLCD4MicroBeads or CD8MicroBeads, mixes well, and hatches at 4~8 DEG C 15min washs cell with 1mLBuffer, is centrifuged (300r/min, 20 DEG C) 10min, discards supernatant 500uLBuffer and is resuspended carefully MS splitter is placed in the magnetic field of MACS separator, is rinsed with 500uLBuffer by born of the same parents, by 500uL cell suspension by dividing It from column, is rinsed splitter repetitive operation 3 times with 500uLBuffer, collects efflux, contain non-CD4+T lymphocyte in efflux Or non-CD8+T lymphocyte, splitter is taken out from separator, with 1000uLBuffer pressure flush splitter, collects outflow Liquid, this (cell viability detection: takes 15uL cell outstanding respectively for CD4+T lymphocyte or CD8+T lymphocyte before and after cell purification Liquid is mixed with isometric trypan blue solution, and the not colored shinny person of microscopically observation is living cells, and the coloring person of swelling is dead cell, Calculate the percentage of living cells in 200 cells).

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 T4 DNA 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 the site SalI, 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 limitation Property restriction endonuclease EcoR I and Xho each 0.5ul of I, 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, be added 5uL electrophoresis sample-adding Buffer (can also pass through be added 0.5mol/L EDTA) terminates reaction, routinely after PCR method amplification hTERT, collect amplified matter with Standby electrophoresis.(ii) it hTERT electrophoresis: takes electrophoresis grade agarose to be made into 10% Ago-Gel with electrophoretic buffer, pours into and sealed Gel casting platform on, plug sample comb, wait be gelled it is solid after envelope band is removed from glue platform, extract comb, be put into added with In the electrophoresis tank of enough electrophoretic buffers, buffer is higher by gel surface about 1mm, is prepared with suitable 10 × sample loading buffer Then sample is added in sample well with pipettor, and does suitable standard control object simultaneously by hTERT digestion sample, connect electricity Pole keeps the hTERT Ghandler motion that faces south dynamic, then under the voltage of 1-10V/cm (80V) gel electrophoresis to being sufficiently separated hTERT segment Apart from when (30min), close power supply.(iii) hTERT purifying is with recycling: hTERT band is separated from agarose: in long wave purple The gel-tape of the segment of hTERT containing target is cut under outer light source and is fitted into bag filter, 2ml running buffer is added into bag filter Liquid is allowed to submerge gel, and empties steam bubble, and bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), is added suitable It measures buffer and bag filter is submerged into (about 6-7mm), power on, 150 volts of electricity are washed, and observe all move to hTERT in the UV lamp Gel out changes direction of an electric field and continues to be powered 1 minute, from buffer is sucked out in bag filter in 1-5ml Eppendorf pipe, adds Entering 1.5 times of volume n-butanols, mixes extracting and remove EB, most high speed 2 minutes on desk centrifuge suck 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 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 hTERT precipitating, abandons supernatant, dry ethyl alcohol is abandoned after being added 70% ethanol washing 2 times, and 50 μ l TE dissolution is added hTERT.In addition, also can be used low melting-point agarose gel method, hTERT filter membrane inserted sheet method etc. by purpose hTERT segment from gel It separates, be 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 110mmol/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 should be larger using surface area and with baffle plate to improve yield 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, 1 500g are centrifuged 10min, and 70% ethyl alcohol of 2mL is added and gently washs precipitating, then of short duration rapid centrifugation, sucks ethyl alcohol, very Sky is 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, inoculation 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；PRMI1640 is supplied to 100%), being placed in 37 DEG C, in volume fraction 5%CO2 incubator, is cultivated 1-2 days, is centrifuged, Supernatant is removed, it is 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) 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 occurs in available flow cytometry analysis cell line, if do not had for system Have, also 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 hTERT integration, expression.(vii) Determined dna sequence: routinely sequenator detects, and shows hTERT gene order.(v) transfect cell DNA in hTERT detection: such as with Immunohistochemical 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 carefully It is intracellular；(vi) mRNA expression product measures: the pcr amplification product of 100 μ l systems is taken, with gel reclaims kit (Takara, day This) recovery product, it takes 2 μ l DNA solutions to dilute 100 times, surveys concentration, remaining DNA and each 10 μ l of upstream and downstream primer are surveyed Sequence.

(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 (1200r/min, 6min), cell is resuspended with 0.5~1ml of frozen stock solution containing dimethyl sulfoxide, carefully Born of the same parents' density is 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；- 70 DEG C, overnight, enters -196 DEG C of liquid nitrogen and freeze It deposits, 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 BamHI (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, and 5uL electricity is added Sample loading buffer (can also be by the way that 0.5mol/L EDTA is added) of swimming terminates reaction in case electrophoresis.(ii) SV40DNA electrophoresis: electricity is taken Swimming grade agarose is made into 10% Ago-Gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs sample Comb removes envelope band from glue platform after being gelled admittedly, extracts comb, be put into the electrophoresis tank added with enough electrophoretic buffers, Buffer is higher by gel surface about 1mm, DNA sample is prepared with suitable 10 × sample loading buffer, then with pipettor by sample It is added in sample well, and does suitable DNA molecular amount standard control object simultaneously, connect electrode, keep the DNA Ghandler motion that faces south dynamic, in 1- Under the voltage of 10V/cm gel electrophoresis to be sufficiently separated DNA fragmentation apart from when, close power supply.(iii) it is separated from agarose About 2600bp SV40 large T antigen DNA: (using long wave ultraviolet light source to prevent DNA from damaging under 300-360nm long wave ultraviolet light source Wound) gel-tape containing target DNA fragments is cut is fitted into bag filter, into bag filter, addition 2ml electrophoretic buffer, is allowed to Gel is submerged, and empties steam bubble, bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), appropriate amount of buffer solution is added Bag filter is submerged into (about 6-7mm), is powered on, 150 volts of electricity are washed, and observe all remove gel to DNA in the UV lamp, are changed Direction of an electric field continues to be powered 1 minute, and from buffer is sucked out in bag filter in 1-5ml Eppendorf pipe, 1.5 times of volumes are added N-butanol mixes extracting and removes EB, and most high speed 2 minutes on desk centrifuge suck upper layer butanol solution, and so repeatedly two It is secondary, isometric phenol chloroform (2) are added from the solution of lower layer speech DNA and is extracted 2 times, and supernatant is transferred in another Eppendorf pipe 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohols is added, stays overnight in 20 DEG C, 12000g, is centrifuged 10 minutes at 4 DEG C, DNA precipitating is obtained, supernatant is abandoned, dry ethyl alcohol is abandoned after being added 70% ethanol washing 2 times, 50 μ l TE dissolving DNAs are added.In addition, also can be used Target DNA fragment is separated from gel, is purified by low melting-point agarose gel method, DNA filter membrane inserted sheet method etc..

(II) connection of SV40 large T antigen DNA and pcDNA3.1 genophore: take 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 bacterium colony is selected with sterile toothpick or disinfection inoculation pin and is inoculated in 5mL In sterile LB culture medium or rich medium (such as super broth or TB super broth culture medium), after overnight incubation, add Into the 2L flask of 500mL culture medium containing LB (containing appropriate antibiotic), then at 37 DEG C of cultures to saturation state (OD₆₀₀≈ 4 is Yield is improved, surface area should be used larger and the flask with baffle plate 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, 1 500g is centrifuged 10min, and 70% ethyl alcohol of 2mL is added and gently washs Precipitating, 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) recon Identification: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competent E.coli, ibid method is with restricted interior Enzyme cutting BamH I carries out digestion, and the identification of 10g/L agarose gel electrophoresis obtains 2 bands of size about 2600bp and 5600bp, preceding Person meets the size of SV40T segment in GenBank.

(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) importing and expansion culture of SV40T/pcDNA3.1: following solutions are prepared in 1.5ml microcentrifugal tube: pipe SV40T/peDNA3.1 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 5-10ml 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 (1 200r/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 blood purification

1, the filling of cleanser

CD4+T cell prepared by the present invention is cleaned with sterile saline, 1000r/min centrifugation, clean after with 1000r/min is centrifuged 5min, takes cell precipitation to be packed into hydrostatic column made of acrylate etc high molecular material, makes Cylindrical column is made up to 4/5 or more in CD4+T cell, and sealing is spare, has separately taken protecting after 100 DEG C dissolve for carrier function Agarose C1-4B physiological saline of the temperature 0.7%, 0.8%, 0.9%, 1.0%, the 1.1% of 42 DEG C, by high concentration to low concentration Successively take 40ml agarose be added cell column in, it is desirable that the agarose being first added be cooled to 37 DEG C become semisolid after just then plus Next time, the layer distributed for the agarose concentration for forming cell column (from import to outlet) from top to bottom from low to high, wherein CD4+T cell, which is fixed in gel, to be play a part of to adsorb HIV.Agar gel is formed by filter opening with the increasing of agarose concentration High and reduce, clarifier entrance agarose concentration is low, and filter opening is just big, and the combination for being conducive to plasma perfusion and cell and HIV is anti- It answers；And exit concentration is high, filter opening is just small, is easy to detention HIV or Large molecular conjugates.Clarifier is combined with a variety of special and non- Special HIV removes composition, 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 absorber 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 absorber 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 absorption 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.Heparin covalent is combined To polyether sulfone surface, the mechanical property of polyether sulfone was not only maintained, but also the anticoagulation function of absorber inner surface can be improved.In acetic acid Covalent immobilisation linoleic acid film on tunica fibrosa, or the linoleic acid for being covalently bound to polyacrylic acid is grafted onto polysulfones film surface, all may be used To have better histocompatbility and anticoagulant effect.With the continuous development of high molecular material and nanotechnology, with mankind's blood The close material of endothelial tube will will appear in the near future.

Three, the preparation of separator

(1) preparation of blood separator

1, preparation principle: 1. haemocyte, bacterium, virus molecular size: visible component (cell) is big in blood of human body It is small are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, and neutrophil leucocyte is about 12 μm, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, and 6-8 μm of small lymphocyte, with red blood cell Approximation, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, and 1~4 micron to 7~8 microns of diameter is differed, the blood of people Platelet average diameter is 2-4 microns, 0.5~1.5 micron thick.The size of bacterium are as follows: the diameter of coccus about 0.75-1.25 μm it Between, for bacillus length about at 2-5 μm, spirillum is about 100-200 μm.The size of virus is with nanometer (nm) for unit [1cm= 10mm, 1mm=1000 μm, 1 μm=1000nm], difference in size is very big between different virus, the smallest gemnivirus such as plant (Geminiviruses) diameter only 18-20nm, maximum animal poxvirus (Poxviruses) size up to 300-450nm × 170-260nm, it is longest if filamentous virus section (Filoviridae) virion size is 80nm × 790-14000nm, majority For the diameter of single virus particle in 100nm or so, AIDS virus is 100-120nm (0.1-0.12 μm).2. AIDS patient's blood Related compounds present in liquid: multinucleate giant cell is (made of gp120 and the CD4+ cell combination of HIV infection cell surface substantially Long-pending HIV infection cell), gp120 cell (there is gp120 on surface but with HIV infection cell existing for individual cells), gene integration (HIV infection initial stage or incubation period are integrated with HIV double-stranded DNA to cell, but cell surface does not have the HIV infection of gp120 thin Born of the same parents), normal white cell (being uninfected by granulocyte, monocyte existing for the individual cells of HIV, lymphocyte), red blood cell, blood it is small Plate, chemical analysis (protein, carbohydrate, lipid, electrolyte etc.), free HIV, bacterium and other microorganisms.3. multicore is big and small Born of the same parents are natural large volume cell；Gp120 cell and gene integration cell can make cell by the immune response of antigen and antibody Agglutination is large volume many cells condensate；Free HIV can be changed into large volume composition by carrier granular/immune response.④ According to above-mentioned 3 points, can prepare can be by individual cells but cannot be by large volume cell or the blood separator of particle.5. selecting The material of the selective adsorption function of apparatus screens out the HIV infection cell in blood through extracorporeal circulation of blood branch of the invention.

2, it the material of blood separator and requirement: with clarifier of the invention, selects poly-vinegar non-woven fabrics, acetate fiber, take off Rouge cotton etc., it is desirable that good biocompatibility, hardly activating complement do not cause inflammatory reaction and leucocyte, blood platelet, blood oxygen point The change of pressure, C3a, C5a.

3, the model of blood separator: the shape of blood separator is prepared into column construction (with acetate fiber or absorbent cotton Equal materials make filter core), the shapes such as flat structure (making filter core with materials such as poly-vinegar non-woven fabrics)；Aperture be prepared into 150~250 μm, The models such as 50~150 μm, 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm, 1~2 μm.

4, the separator of different model, principle the application of principle of blood separator: are selected according to the state of an illness of AIDS patient It is upper first large aperture model to be selected to do pre- sieving, then select the model of smaller aperture due.Severe AIDS patient often occurs serious Opportunistic infections contain different size of composition in blood.Such as containing the fungi of especially big volume, spirillum, tumour cell and its His foreign matter, then selecting aperture is 150~250 μm or 50~150 μm of separator；The monokaryon macrophage of for example sieving HIV infection is thin Born of the same parents, multinucleate giant cell, many cells condensate and the particulate matter for being adsorbed with HIV, and in order to replace vulnerable to HIV infection CD4+ cell then selects 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm etc of model.These types of model is approximate or is less than The volume of single red blood cell, neutrophil leucocyte, small lymphocyte in blood, but red blood cell, neutrophil leucocyte and macrophage tool There is the characteristic of amoeboid movement, micropore more smaller than own vol can be passed through.

(2) 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 in blood of human body The size of (haemocyte) are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, About 12 μm of neutrophil leucocyte, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, small lymphocyte 6- 8 μm, approximate with red blood cell, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, 1~4 micron to 7~8 microns of diameter It differs, the platelet mean diameter of people is 2-4 microns, 0.5~1.5 micron thick.

(2) poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. material: can be selected, it is desirable that good biocompatibility hardly swashs Living 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 immune cell therapy instrument

1, key member: (1) blood separator: for screening out by volume size with multinucleate giant cell or many cells condensate HIV infection cell existing for state, i.e., for removing endoglobar HIV；(2) plasma separator: thin for separating single blood Born of the same parents and blood plasma；(3) plasma purification device: for the 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 immune cell therapy instrument

1, it installs: such as Fig. 1, with sterile working connecting components, including blood separator, plasma separator, plasma purification Device 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 2500 ∪ or 20~30 ∪/kg.

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (15) are connected into venous blood Then pipe opens blood pump (2), blood flow is 100~150ml/min, if Fig. 1 is when arterial blood is through arterial blood line pipe (1), liver When element and blood pump (2) enter blood separator (3), the large volume multinucleate giant cell formed by HIV infection is delayed at blood In liquid/gas separator (3), mononuclear blood cell and blood plasma successively flow into blood through blood outlet (4), blood pump (6) and circulation line (7) It starches separator (8), isolated blood plasma flows into clarifier (11) open at this time through blood plasma pump (9) and blood vessel (10), wait be full of Blood plasma, about 10 minutes, begin releasing blood plasma, flow out through export pipeline (13), synchronous that blood plasma is perfused to clarifier (12), are purifying When blood plasma in device (11) has nearly flowed, perfusion blood plasma is started again at, clarifier (12) begins releasing blood plasma, two parallel connections at this time Clarifier (11) and (12) alternately.Such as indicate Fig. 2 of the internal structure of the blood separator (3) in Fig. 1, blood separation Have many micropores (3) on the tube wall of the inner cavity (2) of device (1), multinucleate giant cell (4) cannot filter micropore (3) and be delayed at interior Chamber (2), so as to be removed, can by micropore (3), the mononuclear blood cell (5) of small size and blood plasma enter exocoel (6), Then (7) outflow is exported, and then separates blood cells and blood plasma through plasma separator shown in FIG. 1 (8).Such as indicate in Fig. 1 Plasma separator (8) internal structure Fig. 3, have many micropores (3) on the tube wall of the inner cavity (2) of plasma separator (1), no There can be the haemocyte of switchable valve outlet (8) outflow by the mononuclear blood cell (4) of micropore (3), entrance is shown in FIG. 1 Haemocyte export pipeline (14)；Plasma separator exocoel (6) can be entered by the blood plasma and its chemical component (5) of micropore (3), so Enter clarifier by blood plasma outflux (7), blood plasma pump shown in FIG. 1 (9) and blood vessel (10).Such as indicate clarifier in Fig. 1 (11) and Fig. 4 of (12) internal structure, when the blood plasma containing HIV (3) enters clarifier (1), HIV (3) therein is fixed CD4+T cell (2) in agar gel is combined into the CD4+T cell conjugates (4) no longer moved down, is not in addition combined The HIV of 100~120nm is again by the bottom agar gel molecular of smaller about 1% concentration of 85nm in aperture due to concentration is higher Screen Out stays, and the purification blood plasma after being adsorbed HIV separates through export pipeline shown in FIG. 1 (13) with plasma separator (8) single Cell converges after export pipeline (14) converges through venous line (15).So purification blood, removing HIV, until being previously set Plasma circulation amount (usually 9L), treatment just end.Entire therapeutic process is controlled by computer, and can detect work at any time Make state, easy to use, automation and safety.

Six, the verifying of AIDS immune cell therapy instrument effect

1, blood separator filters out the verifying of HIV infection cell effect

The present inventor's basic skills according to the invention has done following simple confirmatory experiment: having taken Disease Control and Prevention Center and infection The anticoagulated whole blood several pieces for AIDS (AIDS) patient made a definite diagnosis that sick laboratory biological sample database saves, part phase of fetching respectively Anticoagulated whole blood with abo blood group is mixed into 5, keeps blood volume sufficiently large, then entrusts hospital center of Zhejiang Province blood station proportionately The blood component separation method of part blood transfusion, isolates leucocyte, red blood cell, blood plasma through blood component separation system, takes leucocyte Composition routinely centrifugation inhales abandoning supernatant with suitable physiological saline suspension leukocyte cell pellet and proper ratio is then added Gp120 antibody (Shanghai Guang Rui Biotechnology Co., Ltd), mix 37 DEG C of postposition react 5 minutes, then with aperture be 20~ The blood component separation system of 30um isolates the leucocyte (referred to as big leucocyte) of large volume, again to the leucocyte filtrate of filtration The leucocyte (leucocyte in referred to as) of medium volume is further isolated with the blood component separation system that aperture is 15~25um, Leucocyte in filtrate is the leucocyte (referred to as small white blood cells) of small size, collects large, medium and small leucocyte separation suspension respectively, Conventional centrifugal precipitating, inhales and abandons supernatant, with the large, medium and small leukocyte cell pellet of quantitative liquid shifter difference draws equal amounts, conventional method (mechanical or cell pyrolysis liquid) lytic cell (such as with lysate of the same race, needing dosage equal), takes supernatant, then after centrifugation According to HIV-1p24 antigen detection kit (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known dense Spend the p24 antigen conduct of 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml Control, minimum detection limit are lower than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity 0.5pg/ml~80pg/ml, 15min Interior 450nm measures absorbance (OD), and blank control calibration object absorbance value is not higher than not higher than 0.050,0pg absorbance value 0.100,1000pg/ml absorbance is not less than 1.000, is considered as the positive as absorbance > 0.12, and testing result (table 1) is said Bright, the HIV-p24 content in the leucocyte of AIDS patient different volumes size is different, the HIV-p24 in large, medium and small leucocyte Average content be respectively 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, wherein HIV-p24 is average in large and small leucocyte Content differs 148.6pg/ml, reduces 54.4%；The total content of HIV-P24 is respectively in large, medium and small leucocyte 1375.0pg/ml, 979.9pg/ml, 632.1pg/ml, wherein HIV-p24 total content differs 742.9pg/ in large and small leucocyte Ml reduces 54.3%, through statistical test, t=2.43, p < 0.05.Illustrate the intracorporal large volume leucocyte of AIDS patient or HIV containing high level in the large volume leucocyte formed after the effect of gp120 antibody, can be by implementing skill of the invention Art scheme is removed by separation.

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

2, clarifier (agent) removes the verifying of HIV effect

In order to verify the effect of clarifier (mainly CD4+T cell strain) removes HIV, the present invention devises easy test Method: taking 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draws the CD4+ for being centrifuged (1000r/min, 5min) precipitating respectively T cell is then drawn the heat preservation after 100 DEG C dissolve and is reached in 39~41 DEG C of 0.9% spare agarose C1-4B to 200mm scale To about 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 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 patient are grown, respectively about 10mL, blood plasma injects blood sedimentation tube upper end blank pipe in batches before respectively taking 9mLAIDS to filter, wait flow through The CD4+T cellular layer of blood sedimentation tube lower layer simultaneously after outflow, collects efflux, blood plasma after referred to as AIDS filter out of blood sedimentation tube.Take AIDS Blood plasma and blood plasma after filter before filtering, according 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 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

Above-mentioned simple experiment shows the multinucleate giant cell for being easily fused into large volume by the peripheral blood cells of HIV infection, energy It is filtered out by blood separator；And the HIV in blood plasma, device (CD4+T cell strain, agar gel micropore) removing can be cleaned.Show There is significant removing blood using blood separator and plasma purification device as the AIDS immune cell therapy instrument that critical component is constituted The therapeutic efficiency of the inside and outside inhibition of HIV of cell.

Claims

1. a kind of AIDS immune cell therapy instrument for medical domain, which is characterized in that include blood separator, blood plasma point From device and clarifier, the blood separator can filter out the multinucleate giant cell formed in blood by infected by HIV, blood later Slurry separator can separate the mononuclear blood cell and blood plasma of medium volume, and last absorber can filter out the HIV in blood plasma, only Blood plasma after change is fed back after converging with mononuclear blood cell；The clarifier is will to be formulated in fine jade in conjunction with the CD4+T cell strain of HIV Rouge gel is perfused in hydrostatic column made of high-biocompatibility material later, and the agar gel is formed in clarifier The layer distributed of agarose concentration of the entrance to exit from low to high, the exit of clarifier are provided with sieve.

2. AIDS immune cell therapy instrument according to claim 1, which is characterized in that the blood separator is by inner cavity It being constituted with exocoel, the micropore on next door of the inside and outside chamber by separating inside and outside chamber communicates, and the aperture of the micropore is 1~250 μm, Large volume cell made of single blood cell capable of being passed through but mutually cannot bond or merge by two or more.

3. AIDS immune cell therapy instrument according to claim 2, which is characterized in that the blood separator include with Lower 7 kinds of different specifications, micropore size is respectively 150~250 μm, 50~150 μm, 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm or 1~2 μm.

4. AIDS immune cell therapy instrument according to claim 1 to 3, which is characterized in that select micropore size be 150~250 μm or 50~150 μm of separator separates the fungi, spirillum and/or tumour cell of especially big volume；Select micropore Mononuclear macrophage, the multicore that the separator that aperture is 15~40 μm, 8~15 μm, 5~8 μm or 3~5 μm separates HIV infection are huge Cell, many cells condensate, the particulate matter for being adsorbed with HIV and/or the CD4+ cell vulnerable to HIV infection.

5. AIDS immune cell therapy instrument according to claim 1, which is characterized in that the clarifier is by its exit Sieve, the CD4+T cell strain that plays fixed and molecular sieve agar gel, be formulated in agar gel collectively form molecular sieve Mechanical removal and cellular immunity remove the barrier of HIV.

6. according to claim 1,5 any AIDS immune cell therapy instrument, which is characterized in that the appearance of the clarifier Product is 200~300ml, and entrance top diameter sieve mesh number is 800 mesh, and exit bottom diameter sieve mesh number is 2.0~5.0 mesh, outlet The another setting mesh number in place is 100 aim cell strainers.

7. AIDS immune cell therapy instrument according to claim 6, which is characterized in that the exit bottom diameter 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.

8. AIDS immune cell therapy instrument according to claim 1, which is characterized in that the agarose from low to high The layer distributed of concentration 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.

9. a kind of preparation method of the AIDS immune cell therapy instrument cleanser for medical domain, which is characterized in that with nothing Bacterium physiological saline cleans made CD4+T cell strain, and 1000r/min centrifugation remakes centrifugation after cleaning, sedimentation cell is taken to be packed into In 200ml hydrostatic column made of high-biocompatibility material, cell column is made, heat preservation after melting of learning from else's experience respectively 100 DEG C exists The agarose C1-4B physiological saline of 0.7%, 0.8%, 0.9%, 1.0%, the 1.1% of 42 DEG C, successively by high concentration to low concentration Take 40ml to be added in cell column, the agarose being first added waited to be cooled to after semisolid just then plus next time, make cell column from into Mouth to outlet forms the layer distributed of agarose concentration from low to high and CD4+T cell strain accounts for the 4/5 of potting compound volume, wherein CD4+T cell, which is fixed in gel, to be play a part of to adsorb HIV.

10. any AIDS immune cell therapy instrument of claim 1-8 is preparing the application in purging in vitro device, It is characterized in that, the purging in vitro device includes that one end of arterial blood line pipe (1) goes out through heparin and blood pump (2) with containing waste liquid The blood separator (3) of mouth (5) is connected, and blood separator (3) is through blood outlet (4), blood pump (6), circulation line (7) and blood It starches separator (8) to be connected, the plasma outlet port of plasma separator (8) is through net in parallel with two of blood plasma pump (9) and blood vessel (10) Change device (11) to be connected with clarifier (12), the export pipeline (13) of two clarifiers and the haemocyte of plasma separator (8) export Pipeline (14) converges after converging through venous line (15).