CN106267417A - AIDS therapeutic response device - Google Patents

Abstract

nullThe present invention relates to the AIDS therapeutic response device of biomedical sector，It is characterized in that，With exogenous origin gene integrator change T cell gene structure and prepare immortal cell line and with T cell surface antibody for cell growth activating agent artificial industry preparation can in conjunction with HIV and produce cytokine CD4+ cell，With high-biocompatibility material parcel and make can prevent cell and fragment thereof even HIV leach can for CD4+ cell cytokine production and combine HIV provide place reactor，When the blood plasma of in-vitro separation flows through reactor, HIV and CD4+ cell occurs to combine or enter intracellular and be eliminated，The cytokine simultaneously produced flows out through intercellular substance with blood plasma and feeds back internal，It is achieved thereby that not only remove HIV but also supplement the artificial CD4+T cell replacement therapy will removed outside HIV lead body of cytokine，More feasible than the traditional remedies being confined to the internal HIV of killing but be difficult to and have no side effect.

Description

AIDS therapeutic response device

Technical field

The present invention relates to AIDS therapeutic response device in biomedical sector, be mainly used in external removing and be free on AIDS sufferer The ectoglobular HIV (human immunodeficiency virus) of person, the CD4+T cytokine lacked in meanwhile supplementing HIV sufferers body, reach treatment The purpose of acquired immune deficiency syndrome (AIDS).

Background technology

Acquired immune deficiency syndrome (AIDS) is the biography caused by HIV (human immunodeficiency virus) (Human Immunodeficiency Virus, HIV) Catch an illness, be widely current in the whole world, according to World Health Organization (WHO) (WHO) and the relevant report of UNAIDS, from Since within 1981, the U.S. finds Patient With Aids case, the whole world has 208 countries and regions to receive the serious of acquired immune deficiency syndrome (AIDS) so far Threatening, there are about 40,000,000 people and infected acquired immune deficiency syndrome (AIDS), death toll is more than 20,000,000, and there are about 6000 people every day becomes acquired immune deficiency syndrome (AIDS) sense Dye person, there are about people more than 300 simultaneously every day and dies from acquired immune deficiency syndrome (AIDS).It is in rapid growth period at HIV infected individuals in China, super Cross 1,000,000.Acquired immune deficiency syndrome (AIDS) has become as the great of after tumor, cardiovascular and cerebrovascular disease, tuberculosis, diabetes another facing mankind Infectious disease, it has also become the serious public health of global concern and social problem.

HIV is the retrovirus retrovirus infecting human immune cells, and diameter about 120 nanometer, the most spherical in shape, adventitia is lipoid Peplos (from host cell), and embedding virulent albumen gp120 and gp41, gp41 be transmembrane protein, gp120 is positioned at surface, And be combined by noncovalent interaction with gp41, it is inward from the sphere matrix (Matrix) formed by albumen p17, and albumen p24 The half-cone capsid (Capsid) formed, capsid includes virulent rna gene group, enzyme (reverse transcriptase, intergrase, protease) And other are from the composition of host cell.After HIV enters human body, first by macrophage phagocytic, but HIV quickly changes huge The sour environment at some position in phagocyte, creates the condition of its existence applicable, is not the most killed and the most within it breeds. Because CD4 is the receptor of HIV, so the HIV of breeding passes through its envelope protein gp120 the auxiliary at gp41 in macrophage Under (gp41 plays a part bridge, utilizes self hydrophobic interaction mediate retroviral cyst membrane and cell membrane fusion) enter CD4+ cell (cell, mononuclear phagocyte, dendritic cell etc.), in intracellular rapid propagation, every day produces 10⁹～10¹⁰Virion, and Constantly enter other normal and intracellular duplications of CD4+ of regeneration, manufacture more virus infected cell, make peripheral blood CD4+T Cell sustaining breakdown, minimizing.CD4+T cell is most important immunocyte, and the infected once loses a large amount of CD4+T cell, Whole immune system will suffer deathblow, and the infection to various diseases all loses resistance；It is thin that HIV enters host CD4+ Can also show as after born of the same parents hiding for a long time and not showing clinical symptoms, its geneome RNA reverse transcription becomes double-stranded DNA, with virus Intergrase enters in host cell core, and under the effect of intergrase, double-stranded DNA is incorporated in host cell gene group, integrated Viral DNA be referred to as provirus, the several months of can hiding does not replicates, and causes the AIDS several months to incubation period for many years.? The incubation period of AIDS, HIV mainly breeds in the macrophage and dendritic cell of lymph node, and these cells are internal HIV Depots, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, mitogen, antigen, TNF, IL-2 and lymph Element (LT) can excite HIV provirus gene to replicate at the CD4+T Intracellular transcription infected.After a large amount of propagation, inhibition of HIV Grain constantly discharges from destroyed infection cell and is free on blood, then enters back into other cells and continues course of infection；HIV feels Body can be stimulated after dye to produce envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody.HIV carriers, Chinese mugwort Growing in sick patients serum and measure low-level antiviral neutralizing antibody, wherein AIDS patients level is minimum, and HIV carriers are Height, illustrates the most protected effect of this antibody.But antibody can not be with the viruses contact that retains in mononuclear phagocyte, and HIV Easily there is antigenic variation in envelope protein, original antibody is ineffective, makes neutralizing antibody can not play due effect.Hiding Infective stage, HIV provirus is integrated in host cell gene group, and therefore HIV will not be identified by immune system, so only depending on Cannot be removed by autoimmune function.The critically important reason of another one should be to kill according to antibody, remove antigen Mechanism speculates, after immune antibody is combined with antigen, and immunological effect to be produced, or by activating complement, mediate ADCC effect Dissolve cellular antigen, but HIV is not cellular antigen；Phagocyte phagocytosis is attracted to remove antigen by chemotaxis, But HIV is protected in phagocyte on the contrary, is bred；Antibody has been combined neutralization with antigen, makes to lose appeal, but HIV cannot be killed by immune system, remove, and eventually to recover again appeal after antibody loses neutralization activity.

The antiviral first-line drug that treating AIDS is conventional at present is reverse transcriptase inhibitors, and this medicine can not kill HIV, deposits Individual variation, drug resistance, gland plastochondria toxicity, bone marrow depression, globular anemia, granulocyte and thrombocytopenia, pancreatitis, The multiple toxic and side effects such as hepatic injury, other as hiv protease inhibitor, hiv integrase inhibitor, cytokine, vaccine therapy, Gene therapy and monoclonal antibody passive immunization therapy etc., be all difficult to commonly used because of a variety of causes.There is document report profit The stimulant grown as cell by the monoclonal antibody of T cell surface C D3 molecule, the T that mass propgation HIV sufferers separates After cell, feed back as self therapeutic cells, but HIV breeds also with the cultivation of HIV cell and at endogenous multiplication, increases The feedback of amount T cell result also in the feedback of increment HIV.

CD4+T cell is the one of T lymphocyte, and average life is generally about 7 days, but some T cell particularly warp After immortality chemical conversion cell line (strain) can long-term surviving, infinitely expand.Foreign literature is reported, simian virus 40 (SV40) can make certain There is immortalization in a little human cells.The research such as Poulin DL, Kung AL and Sullivan CS shows, SV40 T antigen gene Importing can accelerate convert cell growth rate, after immortalized cells the most repeatedly passes on, still there is metastable increasing Grow characteristic and functional status, also can retain many phenotypic differentiations of its germinal cell simultaneously.Reilly simian virus large T antigen Vascular smooth muscle cell strain is set up in gene transformation, builds cell model to study the inhibitory action machine of heparin for vascular smooth muscle System.Su etc. utilize the superficial cell strain converted through SV40, build cell model and analyze the synthesis of epithelial cell internal protein Regulating and controlling effect.The superficial cell strain that Miquel etc. convert with SV40, mediates as cell model research laminin，LN 5 Cell adhesion.The prostate epithelial cell strain converted through SV40 such as Webber studies prostate as cell model The physiological function of epithelial cell and secretory function.Racusen etc. grind with through Ad12-SV40 conversion renal cells model Study carefully damage and the disease of proximal convoluted tubule.Hougton etc. convert with SV40 and set up Bone marrow Stromal cell as cell model to grind Studying carefully under certain condition of culture, cell has to adipose cell and the potential of osteoblastic two-way differentiation, studies bone further The mechanism that matter is loose.Foreign study can make cell just keep it is also shown that import exogenous human reverse transcriptase of telomere (hTERT) Often phenotype and differentiating characteristic.The most utilize hTERT to be successfully established the immortalized cell line of some cell, substantially keep dye Colour solid is stable, differentiation is normal, contact inhibition, without the growth characteristics relatively normally such as oncogenicity.In stomatology field, Japanology Person Kamata, Fujita and Fujii transfection hTERT establishes immortal human Gingival Fibroblasts, periodontal cell, pulp cells System and Dental Follicle Cells system, cell population doublings number reaches more than 150 times, and cell all shows original biological characteristics, inducing culture After can express the associated protein of derived cell.The transfection hTERT such as Kitagawa establishes people cementoblast system, cell Multiplication reaches more than 200 times, and cell differentiation mark such as alkali phosphatase, type i collagen etc. are expressed stable.But have no preparation CD4+T Cell is for the report of external treatment acquired immune deficiency syndrome (AIDS).

Summary of the invention

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

The invention aims to provide AIDS therapeutic response device；Another object is intended to provide preparation and the application of reactor Method.

The object of the present invention is achieved like this: optionally infects CD4+T based on HIV by its receptor CD4 molecule thin Born of the same parents and cause CD4+T cell considerable damage and immunodeficiency and pathogenic mechanism, change CD4 by exogenous origin gene integrator The gene structure of+T cell, thus be allowed to be converted into and not only keep archeocyte biological characteristics but also can the most unrestrictedly grow forever OEG cell system, and utilize the stimulant that the peculiar molecular antibody of CD4+T cell surface grows as cell, set up and adapt to vitro human The preparation of work industry can produce again the method for the CD4+T cell of cytokine, the CD4+T cell that will thus prepare in conjunction with HIV It is formulated in cells frozen storing liquid, then prepares further can prevent cell and fragment thereof even with high-biocompatibility material parcel That HIV leaches, can for CD4+T cell cytokine production and combine HIV provide place reactor, in extracorporeal circulation separate When blood plasma flows through reactor, HIV Yu CD4+T cell generation association reaction therein subsequently enters intracellular and removes from blood plasma, The cytokine that CD4+T cell produces simultaneously feeds back internal with blood plasma through intercellular substance outflow reactor, thus realizes the most clear The acquired immune deficiency syndrome (AIDS) artificial CD4+T cell replacement therapy of required cytokine is supplemented again except HIV.

The present invention optionally infects CD4+T cell according to HIV, bioartificial liver treats the preparation method of cellular immortalization And the mechanism such as blood replacing treatment, implement with artificial preparation CD4+T cell and make bioreactor further in order to remove Blood plasma HIV and the treating AIDS new method of supplementary respective fine intracellular cytokine, because the CD4 molecule of CD4+T cell surface is being subject to of HIV Body and become the permissive cell of HIV, HIV, adsorbed HIV can be adsorbed when meeting with HIV and be fixed in reaction with CD4+T cell Device, the bioreactor of preparation can prevent cell and fragment thereof even HIV from leaching, can be CD4+T cell cytokine production and In conjunction with HIV provide place, technical scheme can effectively block new life CD4+T cell again by HIV, can make internal HIV is constantly eliminated and reduces until disappearing, thus reaches the therapeutic purposes of immunologic reconstitution, and cannot kill HIV at present and have Effect blocks from the infection cycle approach of blood plasma to CD4+T cell and somewhat expensive, conventional antiretroviral that medicine side effect is big Therapeutic Method is compared, and the present invention is more economical, it is achieved that manually from internal, HIV is transferred to the external treatment removed newly side Method.

Detailed description of the invention

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

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

Fig. 3 is the internal structure schematic diagram of the AIDS therapeutic response device proposed according to the present invention.

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

In Fig. 2,1 is plasma separator, and 2 is plasma separator inner chamber, and 3 is the micropore on plasma separator inner chamber tube wall, 4 Being the hemocyte that can not pass through micropore (3), 5 is the little molecule blood plasma components that can pass through micropore (3), and 6 is plasma separator exocoel, 7 is blood plasma flow export, and 8 is switchable valve.

In Fig. 3,1 is reactor, and 2 is CD4+T cell, and 3 is the free HIV entering reactor, and 4 is that HIV with CD4+T is thin Born of the same parents' conjugate, 5 is the cytokine that CD4+T cell produces.

Below in conjunction with Fig. 1, Fig. 2 and Fig. 3, embodiment of the present invention are explained in detail.

One, the preparation of AIDS therapeutic response device

1, preparation foundation

(1) prepare according to the permissive cell that CD4+T cell is HIV: after HIV enters human body, (divided containing CD4 by macrophage Son) phagocytosis, but HIV quickly changes the sour environment at macrophage some position interior, creates condition of its existence applicable, no But it is not killed and the most within it breeds.Because CD4 is the receptor of HIV, so the HIV of breeding is by its capsule in macrophage Memebrane protein gp120 and under the auxiliary of gp41 (gp41 plays a part bridge, utilize self hydrophobic interaction mediate retroviral cyst membrane with Cell membrane fusion) enter other CD4+ cells, such as mononuclear phagocyte, dendritic cell etc., particularly CD4+T cell, carefully Intracellular is bred rapidly, and every day produces 10⁹～10¹⁰Virion, and it is intracellular or regeneration constantly to enter other normal CD4+T The intracellular duplication of CD4+T, manufactures more virus infected cell, makes peripheral blood CD4+T cell sustaining breakdown, minimizing.Feel at HIV There is the opportunity being free on blood plasma during dye CD4+T cell, the design Biotherapeutics with CD4+T cell as adherent cell is anti- Answer device, in order to the HIV in adsorption removal in-vitro separation blood plasma, so that internal HIV is constantly eliminated and reduces until disappearing, have Effect blocks newborn CD4+T cell infected by HIV approach again, reaches the therapeutic purposes of immunologic reconstitution.

(2) CD4+T cell can produce cytokine: such as IL-2, IFN-γ, TNF-α, IL-4, IL-6 and IL-10, wherein IL-2 is considered as the main regulatory factors of most important SCIF and dynamic equilibrium, causes CD4+T cell proliferation also Increase the cytotoxicity potential of CD8+T cell, CD4+T cell counting can be made to increase for a long time；The IFN of secretion has promotion to be infected Cause the effect of inflammatory reaction；IL-4 and IL-10 of secretion has the effect of suppression inflammatory reaction；The IL-6 of secretion has participation Inflammatory reaction and the effect of autoimmune disease reaction.HIV be mainly characterized by Progressive symmetric erythrokeratodermia immunodeficiency, including with CD4+T cell is that main immunocyte quantity reduces and dysfunction, and various cytokine secretions reduce or disappear, and cause immunity Afunction.

2, the preparation of reactant

Preparation CD4+T cell (including other CD4+ cells), as the reactant (substrate) of inside reactor absorption HIV.

(1) source of primary lymphocyte: have to sow by way of the Infectious Diseases Lab Sample Storehouse that 1. preserves for scientific research In frozen lymphocyte strain (through inactivation HIV totivirus immunity but be uninfected by the lymphocyte of HIV)；2. blood station is bought fresh dense Contracting leukocyte, then carried out inactivateing the lymphocyte of HIV strain immunity；3. the T lymphocyte series directly bought from businessman (strain)；4. the cord blood lymphocytes cell (through inactivation HIV immunity) preserved for scientific research；The most directly take from because doing other experiments suitable The peripheral blood lymphocyte (for self) of the surplus of the HIV-1 the infected just gathered, uses Histopaque lymphocyte to divide Chaotropic separation mononuclearcell (PBMC).

(2) preparation of CD4+T cell: 1. main agents and instrument: CD4, CD8 immunomagnetic beads (U.S. of Germany sky Ni biology skill Art company limited)；Fluorescein isothiocyanate CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company)；Lymph is thin Born of the same parents separate liquid (Shanghai Heng Xin biochemical reagents company limited)；Ethylenediaminetetraacetic acid (EDTA), 0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；New-born calf serum (Hyclone company)；(Germany is beautiful for MiniMACS magnetic separation system It Ni Bioisystech Co., Ltd)；EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. single core is thin The separation (density－gradient centrifuga－tion method) of born of the same parents (PBMC): aseptic take 20mL blood sample (500IU/mL2mL heparin sodium anticoagulant)；PBS liquid will After hemodilution 2～3 times, fully mixing, 6mL anticoagulant venous blood dropper is slowly superimposed on along tube wall to have added 4mL lymph thin Born of the same parents separate in the 10mL centrifuge tube of liquid horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；It is divided in pipe after Li Xin 3 layers, upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, in upper, middle level Interface has the white cloud and mist layer narrow band based on mononuclearcell to be PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC inserts in another 50mL centrifuge tube, adds 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new life Sanguis Bovis seu Bubali + 2mmol/LEDTA clearly, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add counted under microscope 4 on blood counting chamber Cell (PBMC) sum in individual block plaid.3. CD4+T cell and CD8+T cell is isolated and purified: PBMC cell suspension is divided equally To two 1.5mLEppendorf pipes, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, the every 80uLBuffer of re-suspended cell Containing cell number 10⁷Individual, every 10⁷Individual cell adds 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, at 4～8 DEG C Hatching 15min, uses 1mLBuffer washed cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer weight Outstanding cell, is placed on MS detached dowel in the magnetic field of MACS separator, rinses with 500uLBuffer, is led to by 500uL cell suspension Cross detached dowel, rinse detached dowel repetitive operation 3 times with 500uLBuffer, collect effluent, containing non-CD4+T lymph in effluent Cell or non-CD8+T lymphocyte, take out detached dowel in separator, with 1000uLBuffer pressure flush detached dowel, collects Effluent, this is CD4+T lymphocyte or (the cell viability detection: take 15uL before and after cell purification respectively thin of CD8+T lymphocyte Born of the same parents' suspension mixes with equal-volume trypan blue solution, and the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead Cell, calculates the percentage ratio of living cells in 200 cells).

(3) external industry prepares CD4+T cell

Document is had to report the stimulant utilizing the monoclonal antibody of T cell surface C D3 molecule to grow, great Liang Pei as cell After supporting the T cell that HIV sufferers separates, feed back as self therapeutic cells.But HIV is also with the cultivation of HIV cell Breeding and at endogenous multiplication, the feedback of increment T cell result also in the feedback of increment HIV.The present invention is with SV40 or hTERT immortality Change CD4+T cell, and with CD3 monoclonal antibody for cell growth stimulant, a large amount of amplification CD4+T cells, it is used for preparing reaction Device, in conjunction with blood purification technology, removes HIV in vitro.

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

With the method for hTERT immortalization CD4+T cell it is: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo- HTERT and carrier pLXSNneo, connects hTERT and pLXSNneo separated through PCR amplification, gel electrophoresis with Ligation Mix Digestion products, builds pLXSNneo-hTERT recon, converts DH5a competent cell blue or green with amplification, purification picking resistant to ammonia benzyl Mycin bacterium colony extracting plasmid, imports with lipofection and passes on the T lymphocyte in logarithmic growth in vitro, makes recon with thin The DNA of born of the same parents integrates, and the clone of positive recombinant that amplification culture screen through G418, screening cellular morphology, growth curve, dyeing The test of body caryogram, nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemical staining, thin Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic person same or like with primary cell as hTERT immortality The CD4+T cell changed.

With the method for SV40 immortalization CD4+T cell it is: connect simultaneously through BamHI enzyme action with T4 DNA ligase PcDNA3.1 (-) DNA with PCR amplification, the SV40LTag DNA that separates of agarose gel electrophoresis, structure SV40LTag- PcDNA3.1 (-) recombiant plasmid, convert DH5a competent escherichia coli cell amp-R with amplification, purification picking Bacterium colony extracting plasmid, imports the T lymphocyte of In vitro culture with lipofection, makes the DNA integration of recon and cell, with The cell containing positive recombinant of G418 screening, passes on, amplification culture, screening cellular morphology, cell growth curve, chromosome SV40 big T gene test in the test of caryogram, nude mice tumorigenesis, transfectional cell DNA, mrna expression product measure and determined dna sequence Result meets immortalized cells characteristic person same or like with the primary cell CD4+T cell as SV40 immortalization.

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

(I) extraction of hTERT: (i) enzyme action pClneo-hTERT:hTERT is positioned at the EcoRI of plasmid pCl neo-hTERT And between SalI site, pLXSNneo vector multiple cloning site (MCS) restriction enzyme site Han EcoRI Yu XhoI.Commercially available purchase PCIneo-hTERT plasmid, is dissolved in appropriate ultra-clean H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds restricted enzyme EcoR I and each 0.5ul of Xho I, 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, adds 5uL electrophoresis sample loading buffer (also can be by adding 0.5mol/L EDTA) terminates reaction, routinely after PCR method amplification hTERT, Collect amplified matter in case electrophoresis.(ii) hTERT electrophoresis: power taking swimming level agarose is made into 10% agarose with electrophoretic buffer and coagulates Glue, pours on the gel casting platform sealed, plugs sample comb, removes envelope band, extract after gelling is solid from glue platform Comb, puts in the electrophoresis tank added with enough electrophoretic buffers, and buffer exceeds gel surface about 1mm, with appropriate 10 × Sample loading buffer prepares hTERT enzyme action sample, is then added in sample well by sample with pipettor, and does suitable standard simultaneously Tester, connect electrode, make hTERT face south Ghandler motion move, then under the voltage of 1-10V/cm (80V) gel electrophoresis to enough dividing When the distance of hTERT fragment (30min), close power supply.(iii) hTERT purification and recovery: separate hTERT from agarose Band: under long wave ultraviolet light source, the gel-tape containing target hTERT fragment is cut in loading bag filter, add in bag filter Enter 2ml electrophoretic buffer, be allowed to submergence gel, and empty steam bubble, bag filter level is put into electrophoresis tank (length direction and electrophoresis Parallel), addition appropriate amount of buffer solution, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observe and treat under uviol lamp HTERT all removes gel, changes direction of an electric field and continues energising 1 minute, and from bag filter, sucking-off buffer is in 1-5ml In Eppendorf pipe, adding 1.5 times of volume n-butyl alcohol, EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, sucks Upper strata butanol solution, so repeats secondary, adds equal-volume phenol chloroform (2) and extract 2 times in the solution of lower floor hTERT, Supernatant proceeds to add in another Eppendorf pipe 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C of mistakes Night, 12000g, at 4 DEG C centrifugal 10 minutes, obtain hTERT precipitation, abandon supernatant, abandon dry ethanol after adding 70% washing with alcohol 2 times, add Enter 50 μ l TE and dissolve hTERT.Additionally, can also be used with low melting-point agarose gel method, hTERT filter membrane inserted sheet method etc. by purpose hTERT Fragment separates from gel, is purified.

(II) connection of hTERT Yu pLXSNneo carrier: take the hTERT composition (0.1-5 μ g) of the 9 above-mentioned purification of μ l, 1 μ L10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C incubation 24h, builds pLXSNneo-hTERT recon.

(III) pLXSNneo-hTERT recon purification, expand, identify: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses CaCl₂Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to greatly Most coli strains, operating process is summarized as follows: one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16～20h (such as bacillus coli DH 5 2), or the 16～20h overnight culture that 1ml is fresh, forward to one containing 100mlLB culture medium 1L or In 500ml flask, cultivate about 2～3h (rotary shakers 200～300r/min) in 37 DEG C of violent shakings, survey every 20～30min Amount OD600 value ≈ 0.4, aseptically transfers to one, in ice-cold 50ml polypropylene centrifuge tube, at ice by antibacterial Upper placement 10～20min, is centrifuged with 4000r/min with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) in 4 DEG C 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, uses with 10ml The 0.1mMCaCl of ice pre-cooling₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding turn Head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace of final residual Culture fluid flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, at this point it is possible to rapidly Cell being distributed into aliquot, freezes in liquid nitrogen ,-70 DEG C of storages are standby.(ii) with competence escherichia coli purification, amplification PLXSNneo-hTERT recon: take 200 μ l from every kind of competent cell suspension with the sterile pipette tip of cooling and transfer to nothing In the microcentrifugal tube of bacterium, often pipe adds DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng), rotates gently with mixed Even content, places 30min in ice, centrifuge tube is put into pre-heating to the test tube rack in the circulator bath of 40 DEG C, places 90s～2min, does not shake test tube, quickly transfers in ice bath by pipe, makes cell cooling 1～2min, and every centrifuge tube adds 800 μ LSOC culture medium, is warmed to 37 DEG C with water-bath by culture medium, is then transferred to by pipe on 37 DEG C of shaking tables, and incubation 45min makes antibacterial Recovery, and antibiotic-resistance marker's gene of expression plasmid coding, turn by proper volume (every 90mm flat board is up to 200 μ l) The competent cell changed is transferred to, in the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, flat board is placed in room temperature Absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, after 12～16h, may occur in which bacterium colony.(iii) screen, expand recon: use Sterile toothpick or disinfection inoculation pin select single colony inoculation in LB culture medium aseptic for 5mL or rich medium (such as super meat Soup or TB super broth culture medium) in, after overnight incubation, it is then added to 500mL containing LB culture medium (containing suitable antibiotic) In 2L flask, cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4, for improving yield, should use surface area relatively big and band deflection plate Flask to increase venting quality as far as possible, shaking speed should be greater than 400r/min), in 4 DEG C, 6000g is centrifuged 10min, uses 4mL GTL The resuspended precipitation of solution, and transfer in the high speed centrifugation pipe of a volume >=20mL that (bacterial precipitation can be at-20 DEG C or-70 DEG C Indefinite duration preserves), add the GTE solution containing 25mg/mL lysozyme that 1mL newly joins, resuspended precipitation, place 10min in room temperature, add Enter 10mL and newly join NaOH/SDS solution, and mixing, until liquid becomes homogeneous, limpid and thickness, is placed on ice gently 10min, adds 7.5mL acetic acid solution, is gently mixed with suction pipe until viscosity declines and formed big precipitation, places on ice 10min, in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant in another clean centrifuge tube, if having visible Drift the isopropanol of 0.6 times of volume by several layers of filtered through gauze, can be added, reverse mixing, room temperature places 5～10min, in room Temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs precipitation gently, the ofest short duration the most centrifugal, sucks ethanol, very Empty dry (precipitation can be 4 DEG C of long-term preservations).(iv) qualification of recombiant plasmid and amplification: the single bacterium colony on picking plate, inoculation In 3ml is containing 100ug/ml ampicillin LB culture medium, 37 DEG C, 250r/min shaking table is cultivated, collection culture after 14h, 4 DEG C, 10000r/min be centrifuged 5min, extract in a small amount and purification of Recombinant plasmid by test kit description；Double with EcoRI and HindIII Enzyme action recombiant plasmid reaction system: restricted enzyme each 0.5ul, l0 × buffer 2ul, recombiant plasmid 10ul, add water and supply To 20ul, 37 DEG C of enzyme action 1h.Digestion products carries out 0.8% sepharose electrophoresis, time 30min under 80V voltage conditions, and gel becomes As system is taken pictures；Measure the sequence of recombiant plasmid routinely；Recombiant plasmid, will be containing this matter after enzyme action, order-checking are identified accurately The microbionation of grain in LB culture fluid, amplification cultivation, carry out heavy dose of plasmid by heavy dose of plasmid extraction test kit description Extracting and purifying, ultraviolet spectrophotometer is standby after measuring plasmid concentration and purity.

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

(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5～10nmol/L insulins, 20% hyclone In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, Typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer；15% hyclone (FBS)；1% penicillin And streptomycin；PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, from The heart, removes supernatant, standby.

(VI) pLXSNneo-hTERT recon imports T lymphocyte and amplification culture: make in 1.5ml microcentrifugal tube Standby following solutions: pipe A, is dissolved in pLXSNneo-hTERT recon in 100 μ l serum-free mediums；Pipe B, by 20 μ LLipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, left at room temperature 45min, uses serum-free Culture fluid washs above-mentioned T lymphocyte 2 times.1ml depletion of blood is added in Lipofectamine-pLXSNneo-hTERT mixture Clear culture fluid, mixes gently, drops in above-mentioned T lymphocyte, and (hyclone concentration is to add 1ml serum-free medium 20ml/L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues Continuous cultivation 20h, discards culture fluid, and changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days select living cells After making amplification culture, then strengthen G418 concentration to 800mg L^-1, will can stablize the cell of growth in the G418 environment of high concentration Proceed amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If it is thin Born of the same parents increase slowly, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches Transferring in 75ml culture bottle during to 14ml, every 2-3 week adds 5-10ml fresh culture.Cell was cultivated to 9-10 week (about the 75 generations), still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, and dead cell is (logical less than 10% The scale crossing reading culture vessel judges the increase situation of cell quantity；Differentiate dead cell by trypan blue staining and live thin Born of the same parents.Because normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not enter intracellular；And loss of activity Cell, the permeability of after birth increases, can be dyed blueness by trypan blue, can determine whether as cell the most dead.Method is to draw weekly A certain amount of suspension culture, mixes rearmounted room temperature 5～10 minutes, then makes cell sheet with Trypan Blue agent, 1000 total cellular score of counted under microscope, calculate dead cell and the percentage ratio of non-staining living cells of coloring).Hereafter along with Cultivating increase and the prolongation of incubation time of algebraically, the increase of cell quantity is slack-off, dead cell gets more and more, until cell is not It is further added by, even dissolves, reduce, all dead.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 points Clock, after abandoning supernatant, adds 3ml freezing media (5% DMSO (dimethylsufoxide), 95%FBS) mixing, becomes (cell concentration is about 10 to cell suspending liquid⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, then freeze Exist in-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).

(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: take growth preferably transfection Cell, makes cell suspension, through counting, takes 1.4 × 10 respectively⁴Cell is inoculated in 30 and trains containing 15FBS low sugar DMEM culture medium Support bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation is until cell quantity is decreased obviously, every after cultivating 3 days The cell giving no count every 2 days changes liquid, uses same method to observe transfectional cell at hepato ZYME-SFM serum-free medium In growing state.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and makes After curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof "；(iii) check Chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", then this cell is described System does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA colony in flow cytometry analysis cell line, if do not had Having, there is not tumor feature in explanation cell line yet).Chromosome karyotype analysis method is: by adding preheating in 5mL culture fluid 250ug/ml Colchicine 100ul, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, fixing, film-making, G shows band post analysis karyotype；(iv) Flow cytometry: the cell ratio synthesizing, dividing in detection the 19th continuous cell line Example, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is the result that hTERT integrates, expresses.(vii) Determined dna sequence: sequenator detection routinely, shows hTERT gene order.In (v) transfectional cell DNA hTERT detection: as with Immunohistochemical detection, in the nucleus of hTERT transfection, the visible a large amount of brown particles of dyeing, show that hTERT has been integrated into carefully Intracellular；(vi) mrna expression product measures: takes the pcr amplification product of 100 μ l systems, reclaims test kit (Takara, day with gel This) reclaim product, take 2 μ l DNA solutions and dilute 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer surveys Sequence.

(VIII) hTERT mediates CD4+T cell bank: screen and continue to pass on, amplification culture meets forever after above-mentioned qualification OEG cell characteristic the cell same or like with primary cell, take the difference that growth conditions is good, be in exponential phase Cell from generation to generation, is performing centrifugal separation on (1200r/min, 6min), with the frozen stock solution 0.5～1ml re-suspended cell containing dimethyl sulfoxide, carefully Born of the same parents' density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen and freeze Depositing, the immortalization CD4+T cell bank building biological characteristics stable in this way is standby.

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

(I) extraction of SV40 large T antigen DNA: (i) SV40DNA enzyme action: contain large T antigen gene from commercially available purchase SV40 freeze dried powder or SV40 plasmid, be dissolved in appropriate H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uLH₂O, adds restricted enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, adds 5uL electrophoresis sample loading buffer (also can by add 0.5mol/L EDTA) terminates reaction in case electrophoresis.(ii) SV40DNA electrophoresis: Power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs Sample comb, removes envelope band after gelling is solid from glue platform, extracts comb, put into the electrophoresis tank added with enough electrophoretic buffers In, buffer exceeds gel surface about 1mm, prepares DNA sample with 10 appropriate × sample loading buffer, then with pipettor by sample Product add in sample well, and do suitable DNA molecular amount standard control thing simultaneously, connect electrode, make the DNA Ghandler motion that faces south move, Under the voltage of 1-10V/cm gel, electrophoresis is to when being sufficiently separated the distance of DNA fragmentation, closes power supply.(iii) divide from agarose From about 2600bp SV40 large T antigen DNA: (use long wave ultraviolet light source to prevent DNA under 300-360nm long wave ultraviolet light source Damage) gel-tape containing target DNA fragments is cut in loading bag filter, in bag filter, add 2ml electrophoretic buffer, make Submergence gel, and empty steam bubble, bag filter level put into electrophoresis tank (length direction is parallel with electrophoresis), add appropriate buffering Liquid, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treats that DNA all removes gel, change under uviol lamp Energising 1 minute is continued in changed electric field direction, and from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of bodies Long-pending n-butyl alcohol, mixing extracting removes EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, so repeats two Secondary, in the solution of lower floor speech DNA, add equal-volume phenol chloroform (2) extract 2 times, supernatant proceeds in another Eppendorf pipe Add 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C overnight, 12000g, be centrifuged 10 minutes at 4 DEG C, DNA precipitation, abandon supernatant, abandon dry ethanol after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.Additionally, can also be used with Target DNA fragment is separated from gel, is purified by low melting-point agarose gel method, DNA filter membrane inserted sheet method etc..

(II) connection of SV40 large T antigen DNA and pcDNA3.1 genophore: take 9 μ l above-mentioned DNA composition (0.1-5 μ g), 10 μ l 2 × connection buffer, 1 μ l 10mmol/L ATP, T4 DNA ligase (20～500 sticky end unit) or large intestine bars Bacterium DNA ligase, pcDNA3.1 empty carrier mix, and 15 DEG C of incubation 24h are built into SV40T/pcDNA3.1 recon.

(III) SV40T/pcDNA3.1 recon amplification, separate and identify: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses CaCl₂Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to greatly Most coli strains, operating process is summarized as follows: one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16～20h (such as bacillus coli DH 5 2), or the 16～20h overnight culture that 1ml is fresh, forward to one containing 100mlLB culture medium 1L or In 500ml flask, cultivate about 2～3h (rotary shakers 200～300r/min) in 37 DEG C of violent shakings, survey every 20～30min Amount OD600 value ≈ 0.4, aseptically transfers to one, in ice-cold 50ml polypropylene centrifuge tube, at ice by antibacterial Upper placement 10～20min, is centrifuged with 4000r/min with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) in 4 DEG C 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, uses with 10ml The 0.1mMCaCl of ice pre-cooling₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding turn Head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace of final residual Culture fluid flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, at this point it is possible to rapidly Cell being distributed into aliquot, freezes in liquid nitrogen ,-70 DEG C of storages are standby, hang from every kind of competent cell with the sterile pipette tip of cooling Liquid respectively takes 200 μ l and transfers in aseptic microcentrifugal tube, should add in often pipe DNA or coupled reaction mixture (volume≤ 10 μ l, DNA≤50ng), rotate gently to mix content, ice is placed 30min, centrifuge tube is put into pre-heating to 40 DEG C Circulator bath in test tube rack on, place 90s～2min, do not shake test tube, quickly transfer to pipe, in ice bath, make cell Cooling 1～2min, every centrifuge tube adds 800 μ lSOC culture medium, with water-bath, culture medium is warmed to 37 DEG C, is then transferred to by pipe On 37 DEG C of shaking tables, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, by appropriate bulk The competent cell that long-pending (each 90mm flat board is up to 200 μ l) have converted is transferred to containing 200mmol/LMgSO4 and corresponding antibiotic SOB culture medium on, flat board is placed in room temperature and is absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, may occur in which after 12～16h Bacterium colony.(ii) recon screening, expand and extract: select single colony inoculation in 5mL with sterile toothpick or disinfection inoculation pin In aseptic LB culture medium or rich medium (such as super broth or TB super broth culture medium), after overnight incubation, add In the 500mL 2L flask containing LB culture medium (containing suitable antibiotic), cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4, for Improving yield, surface area should be used relatively big and the flask of band deflection plate is to increase venting quality as far as possible, shaking speed should be greater than 400r/ Min), in 4 DEG C, 6000g is centrifuged 10min, by the 4mL resuspended precipitation of GTL solution, and transfers to the high speed of a volume >=20mL In centrifuge tube (bacterial precipitation can preserve at-20 DEG C or-70 DEG C of indefinite duration), add that 1mL newly joins containing 25mg/mL lysozyme GTE solution, resuspended precipitation, place 10min in room temperature, add 10mL and newly join NaOH/SDS solution, and gently mixing until liquid Body becomes homogeneous, limpid and thickness, places 10min on ice, adds 7.5mL acetic acid solution, is gently mixed until viscous with suction pipe Denseness declines and is formed big precipitation, places 10min on ice, and in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant To the centrifuge tube that another is clean, if there being visible drift that the isopropyl of 0.6 times of volume by several layers of filtered through gauze, can be added Alcohol, reverse mixing, room temperature places 5～10min, and in room temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs gently Precipitation, the ofest short duration the most centrifugal, suck ethanol, and be vacuum dried (precipitation can be 4 DEG C of long-term preservations).(iii) recon Identifying: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence escherichia coli, ibid method is with restricted interior Cutting enzyme BamH I and carry out enzyme action, 10g/L agarose gel electrophoresis is identified, it is thus achieved that 2 bands of size about 2600bp and 5600bp, front Person meets the size of SV40T fragment in GenBank.

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

(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5～10nmol/L insulins, 20% hyclone In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, Typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer；15% hyclone (FBS)；1% penicillin And streptomycin；PRMI1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, centrifugal, Remove supernatant, standby.

(VI) importing of SV40T/pcDNA3.1 and amplification culture: prepare following solutions in 1.5ml microcentrifugal tube: pipe A, is dissolved in SV40T/pcDNA3.1 in 100 μ l serum-free mediums (hyclone concentration is 20ml/L)；Pipe B, by 20 μ LLipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, the underlying 45min of room temperature.Train with serum-free Nutrient solution washs above-mentioned T lymphocyte 2 times.1ml serum-free is added in Lipofectamine-SV40T/pcDNA3.1 mixture Culture fluid, mixes gently, then drops to, in above-mentioned T lymphocyte, be subsequently adding 1ml serum-free medium (hyclone concentration For 20ml/L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), Continuing to cultivate 20h, discard culture fluid, changing concentration is 400mg L^-1G418 culture fluid continue to cultivate, select after 8 days to live thin After born of the same parents make amplification culture, then strengthen G418 concentration to 800mg L^-1, will can stablize the thin of growth in the G418 environment of high concentration Born of the same parents proceed amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If Cell increases slow, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.Work as total amount Reaching to transfer in 75ml culture bottle during 14ml, every 2-3 week adds 5-10ml fresh culture.Cell cultivates about 6-8 week (about the 55 generations), still in logarithmic growth, i.e. incubation time and cell is accelerated in multiplication relation, and dead cell (passes through less than 10% The scale reading culture vessel judges the increase situation of cell quantity；Dead cell and living cells is differentiated by trypan blue staining. Because normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not enter intracellular；And loss of activity is thin Born of the same parents, the permeability of after birth increases, can be dyed blueness by trypan blue, can determine whether as cell the most dead.Method is to draw weekly one Quantitative suspension culture, mixes rearmounted room temperature 5～10 minutes, then makes cell sheet with Trypan Blue agent, aobvious Count 1000 total cellular score under micro mirror, calculate dead cell and the percentage ratio of non-staining living cells of coloring).Hereafter along with training Supporting increase and the prolongation of incubation time of algebraically, the increase of cell quantity is slack-off, dead cell gets more and more, until cell is no longer Increase, even dissolve, reduce, all death.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 points Clock, abandons supernatant, adds 3ml freezing media (5% DMSO (dimethylsufoxide), 95%FBS) mixing, becomes thin (cell concentration is about 10 to born of the same parents' suspension⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen In-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).

(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: take growth preferably transfection Cell, makes cell suspension, through counting, takes 1.4 × 10 respectively⁴Cell is inoculated in 30 and trains containing 15FBS low sugar DMEM culture medium Support bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation is until cell quantity is decreased obviously, every after cultivating 3 days The cell giving no count every 2 days changes liquid, uses same method to observe transfectional cell at hepato ZYME-SFM serum-free medium In growing state.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and makes After curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof "；(iii) check Chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", then this cell is described System does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA colony in flow cytometry analysis cell line, as not having Have, illustrate that tumor feature does not occurs in cell line).Chromosome karyotype analysis method is: by adding preheating in 5mL culture fluid 250ug/ml Colchicine 100ul, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, fixing, film-making, G shows band post analysis karyotype；(iv) Flow cytometry: the cell ratio synthesizing, dividing in detection the 19th continuous cell line Example, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is the result that SV40 large T antigen is integrated, expressed. (viii) determined dna sequence: sequenator detection routinely, shows SV40 large T antigen DNA sequence.In (v) transfectional cell DNA SV40 big T gene test: as with Immunohistochemical detection, dye in the nucleus of SV40 transfection visible a large amount of brown particles, Show that SV40T antigen has been integrated into intracellular；Also T antigen expression in cell, wherein T antigen can be detected by RT-PCR method Primer: forward primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', downstream primer (S4496) 5 '-GAA ATG CCA TCT AGT GAT-3’；The a length of 268bp of amplified production, amplification condition is 94 DEG C, 5min, it may be assumed that (94 DEG C, 1min； 55 DEG C, 1min ,-0.5 DEG C/circulation；72 DEG C, 1min) × 30, (94 DEG C, 30S；40 DEG C, 30S, 72 DEG C, 30S) × 15, expand body System is 50 μ l:[Mg²⁺] 2mmol/L, dNTPs 200 μm ol/L, primer concentration 0.4 μm ol/L, Taq1U, template 5 μ l；Experimental group With the cDNA of the 19th generation cell as template (carry out the synthesis of cDNA the first chain with reference to commercially available cDNA the first chain synthetic agent box, product- 20 DEG C of preservations)；Negative control sets two, does template with the cDNA of sterilized water, primary cell respectively, and positive control is with SV40 DNA (extract SV40 DNA with reference to SDS-proteinase-K pathway for template, because SV40 virus is without peplos, do not use SDS rupture of membranes, take 5 μ l and enter Row 1.5% agarose gel electrophoresis detects, and remaining-20 DEG C save backup)；(vi) mrna expression product measures: T antigen mRNA RT-PCR product checks order: take the amplified production of 100 μ l systems, reclaims test kit (Takara, Japan) with gel and reclaims product, takes 2 μ l DNA solutions dilute 100 times, survey concentration, and remaining DNA and each 10 μ l of upstream and downstream primer checks order.

(VIII) SV40LT gene mediated CD4+T cell bank: screen and continue to pass on, amplification culture accords with after above-mentioned qualification Close immortalized cells characteristic the cell same or like with primary cell, take that growth conditions is good, be in exponential phase The cell of different generations, is performing centrifugal separation on (1200r/min, 6min), resuspended carefully with the frozen stock solution 0.5～1ml containing dimethyl sulfoxide Born of the same parents, cell density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid Nitrogen is frozen, and the CD4+T cell bank building biological characteristics stable in this way is standby.

(4) with the preparation of CD4+T cell identity function granule: can be by CD4 molecule, the CD4 molecule of gene recombinaton and similar The molecule of function by conventional chemical coupling, crosslinking, affine absorption etc. be fixed on carrier make be coated with CD4 molecule Grain, or directly take the replacement CD4+ cell application of intimate granule.It is thin that the CD4+T cell of the present invention represents other CD4+ Born of the same parents, prepare immortalization CD4+T cell including with additive method.

3, the specification of reactor

The CD4+T cell present invention prepared, after cleaning with physiological saline solution, then it is (low to be centrifuged 5min with 1000r/min Speed is centrifuged in short-term), take cell precipitation assembling acrylate etc high-biocompatibility material (identical with plasma separator material) The cylindrical reactor made, to 4/5, then adds cells frozen storing liquid (containing 30% hyclone, 1640 trainings of 12% dimethyl sulfoxide Nutrient solution), make cell concentration reach 80%, shake up gently, sealing, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C Liquid nitrogen cryopreservation is standby.When defrosting uses, need cryopreservation tube be put in 37 DEG C water-baths having been warmed up rapidly, and constantly shake, Make the liquid in pipe melt rapidly, use after then cleaning with physiological saline solution.

Reactor can be infundibulate, and footpath, the end is little, and footpath, top is big, and volume is 200～300ml, imports and exports and is equipped with cell screen cloth, Footpath, import department top sieve number is 800 mesh；Footpath sieve number at the bottom of exit is that (2.5～5.0 mesh are equivalent to 0.1 to 2.0～5.0 mesh ～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, in order to stop the antibacterial of the inhibition of HIV or bigger of 120 nanometers；It is 100 mesh that liquid outlet arranges mesh number The cell strainer of (being equivalent to 4 microns), in order to stop the cell that may leach；Buffering it is provided with between liquid entrance and mesh screen District, the beneficially stability of system circulation.

Two, the preparation of plasma separator

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

2, material: with stable in properties, good biocompatibility, high molecular polymer that permeability is high, it is desirable to activate hardly Complement, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can be by altogether Valency, be grafted, the method such as polymerization improves the structure of material, the regulation microinhomogeneities on surface, hydrophilic, minimizing be to blood coagulation and oxygen Change stress impact thus improve sieving adequacy and biocompatibility, the generation of minimizing complication, as select poly-vinegar nonwoven Cloth, acetate fiber, absorbent cotton etc..

3, type and spec: for the profile of separator, can prepare as filter element with the material such as acetate fiber or absorbent cotton Become column construction, be prepared as the shapes such as flat structure as filter element, by hemocyte to be separated and blood with materials such as poly-vinegar non-woven fabrics The molecular size of slurry composition determines aperture.The plasma separator of the present invention makes hollow fibre type filter, hollow-fiber film diameter Being 270～370 μm, film thickness is 50 μm, and aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm.Blood is only permitted in this hole Slurry filters, but can stop all of cell component.

Three, the application of Biotherapeutics reactor

1, the composition of extracorporeal circulation apparatus and effect

(1) reactor: inclusive reaction agent (CD4+T cell), is used for adsorbing, removing HIV.

(2) plasma separator: separated plasma and hemocyte in extracorporeal circulation apparatus.

(3) sound pulse pressure monitoring: arterial blood pressure monitoring is mainly in order to the stopping state of dynamic monitoring plasma separator micropore, separately Outer in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.When blood flow deficiency, arterial pressure will reduce；When have blood coagulation, Thrombosis, particularly during separator blockage of the micro orifice, arterial pressure will raise；Venous pressure monitoring is used for monitoring pipeline blood backflow Pressure, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow is not enough and venous return syringe needle comes off time, venous pressure Will decline, if the distortion blocking of bloody path return duct or backflow syringe needle occur blocking, venous pressure will raise.

(4) air monitering (Air Detector): be used for monitoring the air bubble of blood pathway, typically use ultrasonic listening Principle, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble, detecting system can drive dynamic, Vein bloody path folder carrys out blocking blood flow, prevents the generation of danger.

Other also include temperature control system, liquid mixing system, off gas system, monitored conductivity system, ultrafiltration monitoring and leakage The parts such as blood monitoring.In a word, on the basis of constitution system of the present invention, it is expected to research and develop automatization's therapeutic equipments further, development For hommization, the personalization for the treatment of, the safety of design and the modularity of operation, automatically monitoring and regulation and control, liquid crystal display, oneself Row judges the micro computer processing systems such as alarm reason and ring off signal.

2, using method

(1) install: with sterile working's connecting components, including plasma separator, reactor and each circulation line.

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

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

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

(5) start: venous line (5) being connected the vein blood vessel of HIV sufferers, then opens blood pump, blood flow is 100～150ml/min, such as Fig. 1, when arterial blood enters plasma separator (4), the blood separated through arterial blood line pipe (1) Slurry arrives reactor (8), blood plasma to be full of, about 10 minutes through circulation line (7) under the effect of blood plasma pump (6), begins paying out Blood plasma, flows out through circulation line (10), synchronizes to irrigate blood plasma to reactor (9), and the blood plasma in reactor (8) has nearly flowed Time, start again at perfusion blood plasma, now reactor (9) begins paying out blood plasma, and two reactors in parallel (8), reactors (9) are handed over For carrying out.So until the plasma circulation amount (usually 9L) being previously set, treatment just ends.If supporting computer program Controlling, whole therapeutic process is by computer control, and can detect duty at any time, and using can convenient, automatization and peace Entirely.Such as Fig. 2, when blood to be separated enters inner chamber (2) of plasma separator (1), through the effect of valve (8), can pass through micro- The little molecule blood plasma components (5) in hole (3) enters the exocoel (6) of separator, then flows out through plasma outlet port (7), and can not pass through The hemocyte (4) of micropore (3) flows out through valve (8).Such as Fig. 3, when the blood plasma containing HIV (3) enters reactor (1), wherein The CD4+T cell (2) that is fixed in reactor of HIV (3) be combined into complex (4) and no longer move down, and CD4+T is thin The cytokine (5) that born of the same parents produce is blended in the blood plasma after having adsorbed HIV and feeds back after the gap outflow reactor of CD4+T cell Internal, meanwhile, footpath, the end screen cloth of reactor exit 2.5～5.0 mesh also can stop the antibacterial of inhibition of HIV or bigger.

Four, the checking of reactor therapeutic efficiency

For understanding effect of CD4+T cell, the present invention devises the method for testing of easy reaction device: take sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draws by centrifugation CD4+T cell that (1000r/min, 5min) precipitate respectively to 200mm scale, Then draw and be incubated after 100 DEG C dissolve at 56 DEG C of 0.9% standby agarose C1-4B, reach the long scale of about 10mm, put erythrocyte sedimentation rate After frame cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but the water of little molecule will not be stoped to become with chemistry The material of part etc separately takes 5 example blood plasma, the most about 10mL of acquired immune deficiency syndrome (AIDS) (AIDS) patient that biological sample bank preserves by d, respectively takes Before 9mLAIDS filter, blood plasma injects blood sedimentation tube upper end blank pipe in batches, the CD4+T cellular layer of blood sedimentation tube lower floor to be flowed through from erythrocyte sedimentation rate After flowing out in pipe, collect effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, according to HIV-1p24 Antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operate, with concentration known 0pg/ml, The p24 antigen of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, minimum Detection limit is less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, and in 15min, 450nm surveys Determining absorbance (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ Ml absorbance is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and testing result (table 1) illustrates, AIDS blood plasma is filtered After crossing easy reaction device, part HIV is adsorbed by CD4+T cell, and after the 1st time filters, HIV total body clearance is 22.84%, warp After 2nd time filters, total body clearance is 35.31%, and after the 3rd time filters, total body clearance is 41.9%.Illustrate along with filtering number of times Increase HIV can constantly be removed, thus reach treat AIDS purpose.

Table 1 AIDS blood plasma filters p24 testing result (pg/ml) before and after easy reaction device

Claims (10)

1. the AIDS therapeutic response device for biomedical sector, it is characterised in that with exogenous origin gene integrator CD4+ cell Genome prepare CD4+ cell immortality cell line, described CD4+ cell immortality cell line can in conjunction with HIV and produce cytokine, It is formulated in the described CD4+ cell immortality cell line of cells frozen storing liquid with high-biocompatibility material parcel again, thus make can Prevent cell and fragment thereof and HIV from leaching, field can be provided for CD4+ cell cytokine production and CD4+ Cell binding HIV again Reactor.

AIDS therapeutic response device the most according to claim 1, it is characterised in that described CD4+ cell includes that CD4+T is thin Born of the same parents, CD4+ mononuclear cell, CD4+ macrophage, CD4+ dendritic cell, made by chemical coupling, crosslinking, affine absorption It is coated with the granule of CD4 molecule, and/or the granule similar to CD4+ cell function.

AIDS therapeutic response device the most according to claim 1, it is characterised in that described CD4+ cell immortality cell line is It is prepared from CD4+ cell surface antibodies for cell growth activating agent.

AIDS therapeutic response device the most according to claim 3, it is characterised in that described CD4+ cell surface antibodies includes CD 3-resisting monoclonal antibody, AntiCD3 McAb/CD28 double antibody.

AIDS therapeutic response device the most according to claim 1, it is characterised in that described reactor and plasma separator phase Even, when the blood plasma separated through plasma separator flows through reactor, HIV Yu CD4+ cell there is combination or entrance intracellular and by clearly Removing, the cytokine simultaneously produced flows out through intercellular substance with blood plasma.

6. according to the arbitrary described AIDS therapeutic response device of claim 1-5, it is characterised in that described exogenous gene includes people Reverse transcriptase of telomere (hTERT), simian virus 40 (SV40).

7. according to the arbitrary described AIDS therapeutic response device of claim 1-6, it is characterised in that the volume of described reactor is 200～300ml, to import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh, footpath sieve number at the bottom of exit Being 2.0～5.0 mesh, liquid outlet arranges the cell strainer that mesh number is 100 mesh, is provided with buffering between liquid entrance and mesh screen District, the beneficially stability of system circulation.

AIDS therapeutic response device the most according to claim 7, it is characterised in that at the bottom of described exit, footpath sieve number is made 7 kinds of different sizes of 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh.

9. claim 1-8 arbitrary described AIDS therapeutic response device application in preparing extracorporeal blood circulating device, it is special Levying and be, described extracorporeal blood circulating device includes arterial blood line pipe (1), separates with blood plasma with blood pump (3) through heparin pump (2) Device (4) is connected, and plasma separator (4) is connected, the most successively through the reactor that blood plasma pump (6) is in parallel with 2 with circulation line (7) It is connected with circulation line (10), venous line (5).

Application the most according to claim 9, it is characterised in that when enabling extracorporeal blood circulating device, arterial blood (whole blood) through arterial blood line pipe (1) enter plasma separator (4), the blood plasma separated under the effect of blood plasma pump (6) through following Endless tube road (7) arrives first reactor (8), blood plasma to be full of, and begins paying out blood plasma, flows out through circulation line (10), simultaneously To second reactor (9) perfusion blood plasma, when the blood plasma in first reactor (8) has nearly flowed, start again at perfusion blood Slurry, now second reactor (9) begins paying out blood plasma, and 2 reactors in parallel alternately, remove HIV and containing cell The hemocyte that the blood plasma of the factor and plasma separator (4) are separated flows out through venous line (5) after converging.