CN106178163A

CN106178163A - Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument

Info

Publication number: CN106178163A
Application number: CN201610541000.1A
Authority: CN
Inventors: 翁炳焕; 李兰娟; 马端; 陆燕; 沈敏
Original assignee: Individual
Current assignee: Shu Lan Hangzhou Hospital Ltd; Womens Hospital of Zhejiang University School of Medicine
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2016-12-07
Anticipated expiration: 2036-07-01
Also published as: CN106178163B

Abstract

nullA kind of acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument for medical domain，It is characterized in that preparation can separated plasma、Mononuclear blood cell and the blood of multinucleated giant cell formed because living away from home HIV and plasma separator，Build SV40LTag pcDNA3.1 () and pLXSNneo hTERT recon，Macrophage immortalization is caused through co-transfection，Cells frozen storing liquid it is formulated in after amplification，Cell concentration is made to reach 80%，Then the depurator that perfusion macromolecular material is made，Macrophage strain therein plays the effect of phagocytosis HIV，Depurator and blood、Plasma separator constitutes crucial extracorporeal circulation apparatus，When blood flows through blood separator，Multinucleated giant cell containing HIV is filtered out，And then the blood plasma separated by plasma separator is when flowing through depurator，HIV therein is by macrophage phagocytic，The mononuclear blood cell that blood plasma after purification is separated with plasma separator feeds back after converging，Thus reach to remove in hemocyte、The purification treatment purpose of outer HIV.

Description

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument

Technical field

The present invention relates to preparation and the application of acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument in medical domain, be mainly used in AIDS The removing of the inside and outside HIV (human immunodeficiency virus) of patient's hemocyte, thus reach to treat 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 (human immunodeficiency virus) is a kind of slow virus (Lentivirus) infecting human immune cells, belongs to reverse transcription sick The one of poison, diameter about 120 nanometer, the most spherical in shape.Outer virionic membrane is lipoid peplos (from host cell), and is embedded with virus Albumen gp120 and gp41.Gp41 is transmembrane protein, and gp120 is positioned at surface, and is combined by noncovalent interaction with gp41.To It is inside the sphere matrix (Matrix) formed by albumen p17, and the half-cone capsid (Capsid) that albumen p24 is formed, capsid In high electron density under Electronic Speculum, include virulent rna gene group, enzyme (reverse transcriptase, intergrase, protease) and other Composition from host cell.

After HIV enters human body, first by macrophage phagocytic, but HIV quickly changes macrophage some position interior Sour environment, creates the condition of its existence applicable, is not the most killed and the most within it breeds.Because CD4 is being subject to of HIV Body, thus in macrophage breeding HIV by its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes self hydrophobic interaction mediate retroviral cyst membrane and cell membrane fusion) (cell, monokaryon are huge to be bitten carefully to enter CD4+ cell Born of the same parents, dendritic cell etc.), in intracellular rapid propagation, produce 10 every day⁹～10¹⁰Virion, and it is normal constantly to enter other And regeneration the intracellular duplication of CD4+, manufacture more virus infected cell, make peripheral blood CD4+T cell sustaining breakdown, subtract Few.Gp120 with the CD4 receptor of HIV be combined can directly activate infected apoptosis, even infected by HIV T cell express Envelope antigen also can start normal T-cell, indirectly causes a large amount of broken of CD4+ cell by the crosslinking of cell surface CD4 molecule Bad, result causes the severe immune deficiency centered by CD4+T cell defect, and patient mainly shows: periphery lymphocyte reduces, T4/T8 proportional arrangement, to phytohaemagglutinin and the loss for reaction of some antigen, delayed allergy declines, and NK cell, huge bites Cytoactive weakens, and the synthesis of the cytokine such as IL2, IFN-γ reduces.CD4+T cell is most important immunocyte, infects Person once loses a large amount of CD4+T cell, and whole immune system will suffer deathblow, and the infection to various diseases is all lost Go resistance.HIV can also show as hiding for a long time and not showing clinical symptoms after entering host's CD4+ cell, its genome RNA reverse transcription becomes double-stranded DNA, enters in host cell core with viral integrase enzyme, and under the effect of intergrase, double-stranded DNA is integrated In host cell gene group, integrated viral DNA is referred to as provirus, and the several months of can hiding does not replicates, and causes The AIDS several months is to incubation period for many years.In the incubation period of AIDS, HIV is mainly in the macrophage and dendritic cell of lymph node Breeding, these cells are internal HIV depots, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, have silk to divide Splitting former, antigen, TNF, IL-2 and lymph element (LT) can excite HIV provirus gene multiple at the CD4+T Intracellular transcription infected System.After a large amount of propagation, inhibition of HIV granule constantly discharges from destroyed infection cell and is free on blood, then enters back into Other cells continue course of infection.

Body can be stimulated after HIV to produce envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody.? Measuring low-level antiviral neutralizing antibody in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers are the highest, and the most protected effect of this antibody is described.But antibody can not be with the virus that retains in mononuclear phagocyte Contacting, and HIV envelope protein easily occurs antigenic variation, original antibody is ineffective, makes neutralizing antibody not play due Effect.In the latent infection stage, HIV provirus is integrated in host cell gene group, and therefore HIV will not be known by immune system Not, so only relying on autoimmune function and cannot being removed.The critically important reason of another one should be to kill according to antibody Go out, remove antigen mechanism speculate, after immune antibody is combined with antigen, if immunological effect to be produced, or pass through activation Complement, mediation ADCC effect dissolves cellular antigen, but HIV is not cellular antigen；Phagocytosis is attracted by chemotaxis Antigen is removed in cell phagocytosis, but HIV is protected in phagocyte on the contrary, breeds；Antibody has been combined neutralization and has made with antigen With, it is allowed to lose appeal, but HIV antigenic structure is changeable, often make antibody be difficult to.

In terms of the treating AIDS method having been used for clinic at present, effect is less preferable: (1) hiv reverse transcriptase suppresses Agent: be only capable of preventing the permissive cell of not yet infected by HIV from infecting, the cell infected do not had therapeutical effect, and toxic and side effects is relatively Many, including gland plastochondria toxicity, bone marrow depression, globular anemia, granulocyte and thrombocytopenia, pancreatitis, intersect resistance in addition The generation of the property of medicine, this kind of medicine is not used alone, and mainly does drug combination, and is easily generated drug resistance mutant, causes under clinical efficacy Fall or inefficacy.(2) hiv protease inhibitor: be easily generated the toxic and side effects such as drug induced hepatic injury, lipid metabolic disorder and drug resistance Property.(3) hiv integrase inhibitor: integrated with host cell DNA by suppression inhibition of HIV DNA and play a role, reverse with HIV Transcriptase inhibitors and protease inhibitor associating use simultaneously and antiviral are had synergism.(4) suppression inhibition of HIV enters suppression Agent: include block gp120 with CD4 be combined, block HIV be combined with accessory receptor, act on gp41 film subunit and act on T pouring Bar cell surface CC-chemokine receptor 5 (CCR5) blocks HIV and enters host cell, but liver and heart are had side effect.(5) Cytokine therapy: be mainly used in regulatory T-cell dynamic equilibrium.(6) HIV vaccine treatment: due to the particularity of HIV, as intrinsic Immunity is not enough to resist HIV and targeting destroys immune system, and virus mutation is rapid, causes the most not yet developing real safety Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, does including antisense technology, RNA bait, RNA Disturb, intrabody, dominant negative mutant, suicide gene etc., but enter the II clinical trial phase stage gene therapy almost No.(8) monoclonal antibody passive immunization therapy: reduce the susceptibility of HIV by lowering CD4+T cell surface CCR5, prolong The progress of slow acquired immune deficiency syndrome (AIDS) and the diffusion of minimizing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10 are applied to HIV person to be had Good toleration and safety, can delay but can not stop virus bounce-back (9) adoptive immunity cell therapy: external in a large number Virus a large amount of amplification can be caused when cultivating CD4+T cell autologous for HIV, increase the CD4+T cell quantity that virus infects, and feed back CD4+T cell may increase the place of internal virus replication, causes virus load to rebound, and on the whole, adoptive immunity is thin Born of the same parents treat without obvious toxic and side effects, also do not obtain satisfied therapeutic effect.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophilic granulocyte in peripheral blood, macrophagocyte Being the mononuclear cell in blood and the macrophage in multiple organ, tissue, both constitute mononuclear phagocyte system.Mononuclear cell Formed by the monocyte precursor Development And Differentiation in bone marrow, account for the 3%-8% of blood middle leukocytes sum, its volume relatively lymph Cell is bigger, and mononuclear cell the most only stops 12-24 hour, subsequently into connective tissue or organ, reaches maturity as huge Phagocyte, macrophage is the differentiation of mononuclear phagocyte system camber, ripe cell type, has stronger phagocytosis merit Can, wandering macrophage is more than mononuclear cell several times, lasts a long time, some months of can surviving in the tissue, the macrophage settled down Having different titles, be Kupffer Cell, be microglia in brain, be osteoclast etc. in bone in liver, it expresses Fc Receptor, C3b receptor and CDl4, play defense function in inherent immunity, is also that the professional antigen participating in adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of HIV (human immunodeficiency virus) (HIV), after HIV enters human body, first suffers huge biting carefully The phagocytosis of born of the same parents, but HIV quickly changes the sour environment at macrophage some position interior, creates condition of its existence applicable, The most it is not killed amount reproduction the most within it to assemble, then HIV is passed to CD4+T cell.

Having document to report, simian virus 40 (SV40) can make some human cell that immortalization occurs.Poulin DL、 The research such as Kung AL and Sullivan CS shows, the importing of SV40LT antigen gene can accelerate to convert the growth rate of cell, After immortalized cells the most repeatedly passes on, still there is metastable multiplication characteristic and functional status, also can retain it simultaneously Many phenotypic differentiations of germinal cell, may be used for the foundation of the some kinds of cell model of human and animal.Also there is document report Road, imports exogenous hTERT and cell can be made to keep normal phenotype and differentiating characteristic, the most utilized hTERT to be successfully established The immortalized cell line of some cell, keeps that chromosome stabilityX, differentiation be normal, contact inhibition, relative without oncogenicity etc. substantially The growth characteristics of " normally ", in stomatology field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes forever Biochemical people's Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number reach 150 times with On, cell all shows original biological characteristics, can express the associated protein of derived cell, Kitagawa after inducing culture Establishing people cementoblast system Deng transfection hTERT, cell multiplication reaches more than 200 times, cell differentiation mark such as alkalescence phosphorus Acid enzyme, type i collagen etc. are expressed stable.Recent research proves, simian virus 40 (SV40) can make some human cell occur Immortalization, can make cell tide over M1 after date and continue survival, Secondary Culture 20-30 generation, and then cell can enter again climacteric (M2 Phase), cell mortality increases and accompanies chromosomal abnormality, and the cell of division gradually decreases, most cell death, and HTERT can maintain stablizing of cell chromosome telomere, so that cell is tided over a crisis the phase (M2 phase), can continue survival, pass on Reach 100-300 generation more than, so setting up immortalized cell line, SV40 with SV40 and hTERT transfected with human in vitro tissue cell simultaneously Cell can be made to tide over the M1 phase, and hTERT can make cell tide over the M2 phase, and than being used alone, one of them cell line set up is more steady Calmly, the life-span is longer.

But have no based on the gene constructed macrophage system of SV40LT and/or hTERT and then expand for acquired immune deficiency syndrome (AIDS) in a large number The report for the treatment of.

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 acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument；Another object is intended to provide AIDS diease occurrence The preparation of thing cellular immunotherapy instrument and application process.

The object of the present invention is achieved like this: it is thin that preparation can filter the large volume containing HIV that many cells are combined into The blood separator of born of the same parents and can separate the plasma separator of mononuclear blood cell and blood plasma, prepares and connects through BamHI enzyme with T4 DNA Cut plasmid SV40LTag DNA and carrier pcDNA3.1 (-) product of DNA and build SV40LTag-pcDNA3.1 (-) restructuring Son and with Ligation Mix connect through EcoR I's and Xho I double digestion plasmid pCIneo-hTERT and carrier pLXSNneo Product and the pLXSNneo-hTERT recon that builds, and then cause its immortalization with liposome co-transfection macrophage, through expanding Being formulated in cells frozen storing liquid after increasing, make cell concentration reach 80%, preventing that then perfusion high-biocompatibility material is made is thin Born of the same parents and fragment thereof leach and can provide the depurator in place for macrophage phagocytic HIV, and wherein macrophage strain is fixed on only Change in device and play phagocytosis HIV and be used, made depurator so that with blood, plasma separator combination additional computer regulatory process and Preparation acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument, the blood separator that the blood in extracorporeal circulation is treated in instrument filters large volume Multinucleated giant cell, be divided into blood plasma and mononuclear blood cell by plasma separator, the purified device of blood plasma is removed after HIV thin with single blood Born of the same parents feed back after converging.

The present invention is made up of blood separator, plasma separator and depurator, wherein the aperture of blood separator be 150～ 250 μm, 50～150 μm, 15～40 μm, 8～15 μm, 5～8 μm, 3～5 μm, 1～2 μm, can filter HIV cell in blood The multinucleated giant cell formed or many cells polymer, plasma separator can separate mononuclear blood cell and the blood plasma of medium volume, blood Cleanser in slurry depurator is macrophage strain, is easy to prolonged cold and saves backup, bite because of huge after being formulated in cells frozen storing liquid The CD4 molecule that cell is expressed is the receptor of HIV (human immunodeficiency virus), has the characteristic of natural phagocytosis HIV, and the present invention simulates macrophage and exists The pattern of internal non-specific phagocytosis, when the blood plasma containing HIV flows through depurator, HIV and macrophage come in contact and quilt Phagocytosis, is detained in depurator therewith, so in the extracorporeal circulation of acquired immune deficiency syndrome (AIDS) plasma purification treatment, containing a large amount of HIV's First large volume cell is filtered by blood separator, and HIV free in blood plasma is cleaned device and removes, the blood plasma after purification with in Isopyknic mononuclear blood cell feeds back after converging, thus removes and be combined in cell surface and/or intracellular HIV and be free on The HIV of blood plasma.The routine that the present invention cannot effectively kill HIV for a long time with the method replacement of the external HIV of filtering is internal degeneration-resistant Transcribe drug treatment.

Detailed description of the invention

Fig. 1 is the application schematic diagram of the acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument proposed according to the present invention.

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

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

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

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

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

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

In Fig. 4,1 is depurator, and 2 is the macrophage strain in depurator；3 is the HIV entering depurator together with blood plasma, The phagosome no longer moved down after the phagocyte phagocytosis that 4 are cleaned in device for HIV, the macrophage that 5 is macrophage generation because of Son.

Below in conjunction with Fig. 1, Fig. 2, Fig. 3 and Fig. 4, the enforcement to the acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument that the present invention proposes Scheme is explained in detail.

One, macrophage strain is built

(1) sorting macrophage

1, primary cell is gathered

(1) single core blood cell is gathered: refer to lymphocyte and the monokaryon separated from blood with density－gradient centrifuga－tion method Macrophage.Concrete grammar is: the White Blood Cells Concentrate buying Blood Center or the Cord blood preserved for scientific research, takes 2mL specimen, 6mL anticoagulation dropper, by hemodilution 2～3 times, is fully slowly superimposed on along tube wall after mixing and has added 4mL and drench by PBS liquid Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tube of bar cell separation liquid；After Li Xin in pipe Being divided into 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, upper, Interface, middle level 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, Draw PBMC and insert in another 50mL centrifuge tube, add 5 times and be centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandon Clear 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new born bovine Serum+2mmol/LEDTA, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add counted under microscope on blood counting chamber Cell (PBMC) sum in 4 block plaid.(2) single core histiocyte is gathered: by Zhejiang University's Tissue Engineering Study platform There is provided.Substantially belonging to the macrophage from spleen, its preparation method is: the 1. acquisition of spleen tissue and transhipment: normally Under committee's approval and patient's informed consent, take the spleen specimen tissue of excision, shred into volume about 1mm immediately³Little Piece of tissue, moves in the sterile sealing bottle equipped with pre-cooling 4 DEG C, is transported to rapidly cell culture chamber.2. spleen tissue cell suspension The preparation of liquid: spleen tissue block moves to aseptic operating platform, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in tissue Blood and ensure the aseptic of tissue.Mechanical lapping spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 In liquid.Filtering to hang with 200 mesh stainless steel filtering nets and be mixed with histiocytic RPMI-1640 liquid, filtrate is spleen tissue cell suspension Liquid (mainly containing erythrocyte, lymphocyte, macrophage etc.).3. erythrocytic cracking in spleen tissue cell suspension: then Centrifugal (1000r/min, 3min) with the washing of RPMI-1640 liquid, to remove cell debris, add Tris-NH4Cl effect 5min, Splitting erythrocyte, Quick spin (1000r/min, 3min), remove the splitting erythrocyte fragment in supernatant, PBS washing centrifugal 3 Secondary, centrifugal 1 time of RPMI-1640 washing, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, Now, main containing spleen tissue macrophage and lymphocyte in suspension.4. the adhere-wall culture of spleen tissue macrophage: will Aforementioned suspension is as cultivating cell stock solution, and Trypan Blue judges vigor and counts, and adjusts cell concentration with RPMI-1640 liquid For (3～5) × 10⁶/ L, will adjust the cell suspension inoculation of concentration in glass culture bottle, and condition of culture is 37 DEG C, 50mL/ LCO₂, 100% humidity, cultivate 2～3h respectively, under phase contrast microscope, observe form.The digestion of adherent spleen tissue macrophage: The digestion of adherent spleen tissue macrophage: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, gained Cell suspension washing is centrifugal (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or hands Postoperative discarded specimen extracts preparation, such as peritoneal cavity liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa etc..(3) gather Amniotic fluid, villus cell: attached hospital for obstetrics and gynaecology of Zhejiang University reproduction genetic laboratory is standby.The approval of reason committee and patient Under informed consent, treating excess syndrome tests the residue amniotic fluid after diagnosis report, villus cell, selects exponential phase cell to continue to employ.

2, sorting attached cell (the preliminary adherent sorting of macrophage)

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

3, sorting cd4 cell

Employing immunomagnetic beads method sorting cd4 cell: 1. main agents and instrument: (Germany U.S. sky Ni is biological for CD4 immunomagnetic beads Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany).2. cd4 cell immunity Magnetic bead sorting method: cell suspension is divided equally to two 1.5mLEppendorf pipes, centrifugal (300r/min, 20 DEG C) 10min, discards Supernatant, the every 80uLBuffer of re-suspended cell contains cell number 10⁷Individual, every 10⁷Individual cell add 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, and hatches 15min at 4～8 DEG C, uses 1mLBuffer washed cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer re-suspended cell, MS detached dowel is placed in the magnetic field of MACS separator, with 500uLBuffer rinses, and by 500uL cell suspension by detached dowel, rinses detached dowel repetitive operation 3 times with 500uLBuffer, Collect effluent, containing non-cd4 cell in effluent, in separator, take out detached dowel, divide with 1000uLBuffer pressure flush From post, collecting effluent, this is that (cell viability detects cd4 cell: takes 15uL cell suspension before and after cell purification respectively and waits body Long-pending trypan blue solution mixing, the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, calculates 200 The percentage ratio of living cells in individual cell).The cell now sorted is mainly macrophage.

4, sorting CDl4 cell (sorting macrophage)

CDl4 is mononuclear cell and the distinctive surface marker of macrophage, if in theory from single core histiocyte, sheep Sort in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, then gained Cell includes mononuclear cell and macrophage；But because the mononuclear cell life-span is short, only survives 1 day in peripheral blood and can not show a candle to huge biting Cell is prone to adherent growth, so the most substantially removing in the cell attachment of the present invention is cultivated, the cell sorted out is essentially Macrophage.

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

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

(2) SV40LT/pcDNA3.1 and hTERT/pLXSNneo recon is built

1, SV40LT and hTERT gene is extracted

(1) extraction of SV40LT and hTERT: (I) enzyme action SV40LT and hTERT: 1. enzyme action SV40LT: contain from commercially available purchase There are SV40 freeze dried powder or the SV40 plasmid of large T antigen gene, are dissolved in appropriate H₂In O or TE buffer, add 2uL10 × enzyme Cut buffer, 18uL H₂O and restricted enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, go out Live enzyme, add 5uL electrophoresis sample loading buffer (also can by add 0.5mol/L EDTA) and terminate reaction in case electrophoresis.2. enzyme action HTERT:hTERT between EcoRI and the SalI site of plasmid pClneo-hTERT, pLXSNneo vector multiple cloning site (MCS) restriction enzyme site Han EcoRI Yu XhoI.From commercially available purchase pCIneo-hTERT plasmid, it is dissolved in appropriate ultra-clean H₂O or TE In 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, adding 5uL electrophoresis sample loading buffer (also can be by adding 0.5mol/L EDTA) terminate reaction, routinely after PCR method amplification hTERT, collect amplified matter in case electrophoresis.(II) SV40LT and hTERT electricity Swimming: 1. SV40LT (SV40DNA) electrophoresis: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours into On the gel casting platform sealed, plug sample comb, after gelling is solid, from glue platform, removes envelope band, extracts comb, put into In electrophoresis tank added with enough electrophoretic buffers, buffer exceeds gel surface about 1mm, by 10 appropriate × sample loading buffer system Standby DNA sample, then adds sample in sample well with pipettor, and does suitable DNA molecular amount standard control thing simultaneously, connect Energising pole, makes the DNA Ghandler motion that faces south move, and under the voltage of 1-10V/cm gel, electrophoresis is to when being sufficiently separated the distance of DNA fragmentation, closes Close power supply.2. hTERT electrophoresis: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours into and has sealed On gel casting platform, plug sample comb, after gelling is solid, from glue platform, removes envelope band, extracts comb, put into added with foot In the electrophoresis tank of enough electrophoretic buffers, buffer exceeds gel surface about 1mm, prepares with 10 appropriate × sample loading buffer HTERT enzyme action sample, then adds sample in sample well with pipettor, and does suitable standard control thing simultaneously, connects electricity Pole, make hTERT face south Ghandler motion move, under the voltage of 1-10V/cm (80V) gel, electrophoresis is to the distance being sufficiently separated hTERT fragment Time (30min), close power supply.(III) SV40LT and hTERT purification and recovery: 1. SV40LT purification and recovery: from agarose Isolate about 2600bp SV40 large T antigen DNA: under 300-360nm long wave ultraviolet light source (use long wave ultraviolet light source in case Only DNA damage) gel-tape containing target DNA fragments is cut in loading bag filter, in bag filter, add 2ml running buffer Liquid, is allowed to submergence gel, and empties steam bubble, and bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), adds suitable Amount buffer, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treat that DNA all removes under uviol lamp Gel, changes direction of an electric field and continues energising 1 minute, and from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of volume n-butyl alcohol, mixing extracting removes EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, as This repeats secondary, adds equal-volume phenol chloroform (2) and extract 2 times in the solution of lower floor DNA, and supernatant is transferred to another 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol of addition in Eppendorf pipe, in 20 DEG C overnight, 12000g, 4 DEG C Under be centrifuged 10 minutes, obtain DNA precipitation, abandon supernatant, after adding 70% washing with alcohol 2 times, abandon dry ethanol, add 50 μ l TE and dissolve DNA.Additionally, can also be used with low melting-point agarose gel method, DNA filter membrane inserted sheet method etc. target DNA fragment is separated from gel, pure Dissolve.2. hTERT purification and recovery: separate hTERT band from agarose: target will be contained under long wave ultraviolet light source The gel-tape of hTERT fragment cuts in loading bag filter, adds 2ml electrophoretic buffer, be allowed to submergence gel in bag filter, And empty steam bubble, bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), adds appropriate amount of buffer solution by bag filter Submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treats that hTERT all removes gel, change electric field side under uviol lamp To continuing energising 1 minute, from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of positive fourths of volume Alcohol, mixing extracting removes EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, so repeats secondary, from Adding equal-volume phenol chloroform (2) in the solution of lower floor hTERT to extract 2 times, 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 overnight, 12000g, it is centrifuged 10 minutes at 4 DEG C, obtains hTERT Precipitation, abandons supernatant, abandons dry ethanol after adding 70% washing with alcohol 2 times, adds 50 μ l TE and dissolves hTERT.Additionally, can also be used with low Purpose hTERT fragment is separated from gel, is purified by melting agarose gel method, hTERT filter membrane inserted sheet method etc..

2, SV40LT/pcDNA3.1 recon is built

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 e. coli dna ligase, the mixing of pcDNA3.1 empty carrier, 15 DEG C of incubations 24h, is built into SV40LT/pcDNA3.1 recon.

3, hTERT/pLXSNneo recon is built

Take the hTERT composition (0.1-5 μ g) of the 9 above-mentioned purification of μ l, 1 μ l 10mmol/L ATP, 10ul Ligation Mix Or the mixing of e. coli dna ligase, 2ul pLXSNneo empty carrier, 15 DEG C of incubation 24h, build pLXSNneo-hTERT restructuring Son.

4, purification, expand, identify SV40LT/pcDNA3.1 and hTERT/pLXSNneo recon

(I) SV40LT/pcDNA3.1 recon amplification, separate and identify: the 1. preparation of E. coli competent: its base This method 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 After upper placement 10～20min, in 4 DEG C with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) with 4000r/min from Heart 10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, with 10ml Ice-cold 0.1mMCaCl₂Resuspended every part of precipitation, is put on ice, (or its corresponding with SorvallGS3 rotary head in 4 DEG C Rotary 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 Amount 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 fast Cell is distributed into aliquot by speed, freezes in liquid nitrogen, and-70 DEG C of storages are standby, with the sterile pipette tip of cooling from every kind of competent cell Suspension respectively takes 200 μ l and transfers in aseptic microcentrifugal tube, often pipe add 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 following of pre-heating to 40 DEG C On test tube rack in ring water-bath, place 90s～2min, do not shake test tube, quickly pipe is transferred in ice bath, make cell cool down 1～2min, every centrifuge tube adds 800 μ lSOC culture medium, with water-bath, culture medium is warmed to 37 DEG C, then pipe is transferred to 37 DEG C On shaking table, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, and proper volume is (every Individual 90mm flat board is up to 200 μ l) competent cell that converted transfers to containing 200mmol/LMgSO4 and the SOB of corresponding antibiotic In culture medium, flat board is placed in room temperature and is absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, after 12～16h, may occur in which bacterium colony. 2. recon screening, expand and extract: select single colony inoculation in LB aseptic for 5mL with sterile toothpick or disinfection inoculation pin In culture medium or rich medium (such as super broth or TB super broth culture medium), after overnight incubation, it is then added to 500mL and contains In the 2L flask of 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 in the high speed centrifugation pipe of a volume >=20mL (bacterial precipitation can preserve at-20 DEG C or-70 DEG C of indefinite duration), adds the GTE solution containing 25mg/mL lysozyme that 1mL newly joins, Resuspended precipitation, places 10min in room temperature, adds 10mL and newly joins NaOH/SDS solution, and mixes until liquid becomes equal gently One, limpid and thickness, places 10min on ice, adds 7.5mL acetic acid solution, is gently mixed with suction pipe until viscosity declines And form big precipitation, and 10min is placed on ice, in 4 DEG C, 20 000g are centrifuged 10min, and supernatant is poured into gently In clean centrifuge tube, if there being visible drift that the isopropanol of 0.6 times of volume by several layers of filtered through gauze, can be added, reverse Mixing, room temperature places 5～10min, and in room temperature, 1 500g is centrifuged 10min, adds 2mL70% ethanol and washs precipitation gently, then Of short duration the most centrifugal, suck ethanol, and be vacuum dried (precipitation can be 4 DEG C of long-term preservations).3. the qualification of recon: above-mentioned from sense By the DNA (containing SV40T/pcDNA3.1) extracted in state escherichia coli, ibid method restricted enzyme BamH I carries out enzyme action, 10g/L agarose gel electrophoresis is identified, it is thus achieved that 2 bands of size about 2600bp and 5600bp, the former meets in GenBank The size of SV40T fragment.(II) hTERT/pLXSNneo recon purification, expand, identify: the 1. system of E. coli competent Standby: its basic skills is ice-cold CaCl₂Or multiple divalent cation etc. processes antibacterial, feed them into competence and turned Change, use CaCl₂Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is fitted For most of coli strains, operating process is summarized as follows: picking one from 37 DEG C of fresh plate cultivating 16～20h Single bacterium colony (such as bacillus coli DH 5 2), or the 16～20h overnight culture that 1ml is fresh, forward one to containing 100mlLB culture medium 1L or 500ml flask in, cultivate about 2～3h (rotary shakers 200～300r/min) in 37 DEG C of violent shakings, every 20～ 30min measures OD600 value ≈ 0.4, aseptically antibacterial is transferred to one, ice-cold 50ml polypropylene centrifuge tube In, place 10～20min on ice, in 4 DEG C with Sorvall6S2 rotary head (or the rotary head matched with its centrifuge tube) with 4000r/ Min is centrifuged 10min, to reclaim cell, and culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, With the ice-cold 0.1mMCaCl of 10ml₂Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or and its Corresponding rotary head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the most residual The trace culture fluid stayed flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling₂Resuspended every part is sunk calmly, rapidly will Cell is distributed into aliquot, freezes in liquid nitrogen, and-70 DEG C of storages are standby.2. with competence escherichia coli purification and amplification hTERT/ PLXSNneo recon: take 200 μ l from every kind of competent cell suspension respectively with the sterile pipette tip of cooling and transfer to aseptic In microcentrifugal tube, often pipe adds DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng), rotate 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/LMgS04 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.3. screen, expand recon: with aseptic Toothpick or disinfection inoculation pin select single colony inoculation in LB culture medium aseptic for 5mL or rich medium (such as super broth or TB super broth culture medium) in, after overnight incubation, it is then added to the 500mL 2L containing LB culture medium (containing suitable antibiotic) and burns In Ping, cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4, for improving yield, should use surface area relatively big and the burning of band deflection plate Bottle is to increase venting quality as far as possible, and shaking speed should be greater than 400r/min), in 4 DEG C, 6000g is centrifuged 10min, with 4mL GTL solution Resuspended precipitation, and transfer in the high speed centrifugation pipe of a volume >=20mL that (bacterial precipitation can be unlimited at-20 DEG C or-70 DEG C Phase preserves), add the GTE solution containing 25mg/mL lysozyme that 1mL newly joins, resuspended precipitation, place 10min in room temperature, add 10mL newly joins NaOH/SDS solution, and mixing, until liquid becomes homogeneous, limpid and thickness, places 10min on ice gently, Add 7.5mL acetic acid solution, be gently mixed with suction pipe until viscosity declines and formed big precipitation, place 10min on ice, In 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant in another clean centrifuge tube, if there being visible floating Thing by several layers of filtered through gauze, can add the isopropanol of 0.6 times of volume, reverse mixing, and room temperature places 5～10min, in room temperature, 1 500g is centrifuged 10min, adds 2mL70% ethanol and washs precipitation gently, the ofest short duration the most centrifugal, sucks ethanol, and vacuum is done Dry (precipitation can be 4 DEG C of long-term preservations).4. the qualification of recombiant plasmid and amplification: the single bacterium colony on picking plate, is inoculated in 3ml and contains In 100ug/ml ampicillin LB culture medium, 37 DEG C, 250r/min shaking table is cultivated, collection culture after 14h, 4 DEG C, 10000r/min is centrifuged 5min, extracts in a small amount and purification of Recombinant plasmid by test kit description；With the double enzyme of EcoRI and HindIII Cut recombiant plasmid reaction system: each 0.5ul of restricted enzyme, 10 × buffer 2ul, recombiant plasmid 10ul, adding water complements to 20ul, 37 DEG C of enzyme action 1h.Digestion products carries out 0.8% sepharose electrophoresis, time 30min, gel imaging under 80V voltage conditions System is taken pictures；Measure the sequence of recombiant plasmid routinely；Recombiant plasmid, will be containing this plasmid after enzyme action, order-checking are identified accurately Microbionation in LB culture fluid, amplification cultivation, carry out heavy dose of plasmid by heavy dose of plasmid extraction test kit description and take out Purification, ultraviolet spectrophotometer is standby after measuring plasmid concentration and purity.

(3) macrophage strain is built

1, co-transfection SV40LT and hTERT causes macrophage immortalization

Recon SV40LT/pcDNA3.1 and hTERT/pLXSNneo is imported macrophage, builds macrophage strain, and Row amplification culture, method particularly includes: in 1.5ml microcentrifugal tube, prepare following solutions: take A pipe, by SV40LT/pcDNA3.1 It is dissolved in 50 μ l serum-free mediums；Take B pipe, hTERT/pLXSNneo is dissolved in 50 μ l serum-free mediums；Then C is taken 20 μ l liposome Lipofectamine are dissolved in 80 μ l serum-free mediums by pipe, by A pipe, B pipe and the mixing of C pipe, in room 45min is stood under conditions of temperature.Above-mentioned T lymphocyte is washed 2 times with serum-free medium.Exist the most respectively In Lipofectamine-SV40LT/pcDNA3.1 and Lipofectamine-hTERT/pLXSNneo mixture add 1ml without Serum free culture system liquid, mixes gently, then drops to, in above-mentioned T lymphocyte, be subsequently adding 1ml serum-free medium (hyclone Concentration is 20ml/L), at CO₂Incubator cultivates 10h, and sucking-off transfection liquid, (hyclone concentration is to add 4ml complete culture solution 20%), continuing to cultivate 20h, discard culture fluid, changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days select Select after living cells makees amplification culture, then strengthen G418 concentration to 800mg L^-1, raw by stablizing in the G418 environment of high concentration Long cell proceeds amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that macrophage significantly increases, occur assembling now As.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 and changes Liquid.Transfer in 75ml culture bottle when total amount reaches 14ml, every 2-3 week adds 5-10ml fresh culture.Cell is cultivated extremely 15-20 week (the about the 135th generation), still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, dead Cell (judges the increase situation of cell quantity less than 10% by reading the scale of culture vessel；Reflected by trypan blue staining Other dead cell and living cells.Because normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not enter born of the same parents In；And the cell of loss of activity, the permeability of after birth increases, and can be dyed blueness by trypan blue, can determine whether as cell the most dead. Method is to draw weekly a certain amount of suspension culture, mixes rearmounted room temperature 5～10 minutes with Trypan Blue agent, then makes Become cell sheet, under the microscope 1000 total cellular score of counting, calculate the dead cell and the hundred of non-staining living cells of coloring Proportion by subtraction).Hereafter along with increase and the prolongation of incubation time of cultivation algebraically, the increase of cell quantity is slack-off, dead cell more comes The most, until cell is not further added by, even dissolve, reduce, all dead.When total amount reaches 45ml, put in 50ml centrifuge tube, Centrifugal 1500 turns, 10 minutes, abandon supernatant, add 3ml freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then Put-70 DEG C of 2h, 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).With this Build immortalization macrophage strain (being).

2, the biological characteristics of macrophage strain is identified

1. form under observation of cell mirror: visible macrophage slurry is abundant, irregularly, flexible under liquid；2. observe thin Intracellular growth curve: take the preferable transfectional cell of growth, make cell suspension, through counting, take 1.4 × 10 respectively⁴Cell is inoculated in 30 contain 15 FBS low sugar DMEM culture medium culturing bottles, take 2 bottles of cells every day and count, calculate average, Continuous Observation until Cell quantity is decreased obviously, and the cell giving no count every 2 days after cultivating 3 days changes liquid, uses same method to observe transfectional cell Growing state in hepato ZYME-SFM serum-free medium, result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), be depicted on semi-logarithmic scale make after curve the growth curve of this cell, immortalized cell line is in typically " S " feature or " arched roof " are formed；3. chromosome is checked: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", or identical with the caryogram of primary cell, then illustrate that this cell line does not occur the vicious transformation (simultaneously can With whether flow cytometry analysis cell line occurs abnormal DNA colony, if it did not, tumor does not occurs in explanation cell line yet Feature)；4. nude mice tumorigenesis test: by the cell of hTERT and SV40LT immortalization with 3 × 10⁷Inoculation nude mice dorsal sc, 2 After Yue, 4 nude mices are showed no tumor and are formed, it was demonstrated that this cell is non-malign cells；5. hTERT and SV40 in transfectional cell DNA Big T gene test: as with Immunohistochemical detection, dye in the nucleus of hTERT and SV40 transfection visible a large amount of browns Grain, shows that hTERT or SV40LT antigen has been integrated into intracellular；Also the expression in cell of the T antigen can be detected by RT-PCR method, The wherein primer of T antigen: 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, I.e.: (94 DEG C, 1min；55 DEG C, 1min ,-0.5 DEG C/circulation；72 DEG C, 1min) × 30, (94 DEG C, 30S；40 DEG C, 30S, 72 DEG C, 30S) × 15, amplification system is 50 μ l:[Mg²⁺] 2mmol/L, dNTPs200 μm ol/L, primer concentration 0.4 μm ol/L, Taq 1U, Template 5 μ l；Experimental group (carries out cDNA with reference to commercially available cDNA the first chain synthetic agent box with the cDNA of the 19th generation cell as template One chain synthesis, product-20 DEG C preservation)；Negative control sets two, does template with the cDNA of sterilized water, primary cell respectively, positive Comparison (is extracted SV40 DNA with reference to SDS-proteinase-K pathway with SV40 DNA as template, because SV40 virus is without peplos, is not used SDS rupture of membranes, takes 5 μ l and carries out 1.5% agarose gel electrophoresis detection, and remaining-20 DEG C save backup)；6. mrna expression product is surveyed Fixed: T antigen mRNA RT-PCR product checks order: takes the amplified production 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.7. Flow cytometry: the cell proportion synthesizing, dividing in detection the 19th continuous cell line, if its multiplication capacity is obvious Strengthening than not building the normal cell being, explanation is the result that hTERT and SV40 large T antigen is integrated, expressed.8. determined dna sequence: Sequenator detection routinely, shows hTERT and SV40 large T antigen gene order.In a word, the spy that the strain of immortalization macrophage has Property be: 1. macrophage transfection SV40LT and hTERT gene thus can cultivate by Long Term Passages；2. the chromosome core of macrophage Type is diploid, i.e. " 46, XX " or " 46, XY "；3. the growth curve of macrophage is such as with incubation time as transverse axis, cell quantity For the longitudinal axis, in " S " feature or " arched roof " is formed in hepato ZYME-SFM serum-free medium；4. cell can not be at soft fine jade Growth in fat (forms clone)；5. cell is non-malign cells, nude mice tumorigenesis negative；6. the DNA of cell integrates simultaneously SV40LT and hTERT gene, expresses the mRNA product of SV40LT and hTERT gene；7. in-vitro cultivation 15-20 week is (about 135th generation) still in exponential phase；8. can the most frozen, Long Term Passages cultivation.

3, the function of macrophage strain is detected

Macrophage strain being inoculated in 96 well culture plates, observation of cell growing state, after 7-10 days, cell clone grows When area reaches 1/10 cell hole, go culture supernatant, the culture hole that growth selection is in good condition, labelling cell strain under microscope The position of growth, size, use aseptic rifle head to draw cell clone to the new cultivation having complete medium in the position of mark In hole, doubling dilution counts hole to below the most successively, 37 DEG C, cultivates about one week, basis of microscopic observation in 5%CO2 incubator Cell growth status, when cell clone covers with to hole floor space more than 1/10, takes cell or culture supernatant detection macrophage The function of strain.Including: 1. macrophage strain phagocytosis antibacterial Function detection: macrophage is hanged with staphylococcus or Candida albicans Liquid mixing incubation, smear, fixing, serge blue liquid dyes, and in oil Microscopic observation phagocytosis situation, counts phagocytosis antibacterial and does not gulps down Bite the macrophage number ratio of antibacterial, to swallow antibacterial function strong macrophage alternately positive clone strain.The hugest bite thin Born of the same parents' strain phagocytosis HIV Function detection: the blood plasma taking acquired immune deficiency syndrome (AIDS) (AIDS) patient that Disease Control and Prevention Center preserves mixes training with macrophage strain After Yanging, separate cell strain, PBS 3 times, measure the phagocyte strain through cracking and swallow the function of HIV, with specific reference 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.Concrete test kit description of pressing operates.The hugest Phagocyte strain produce macrophage cytokines detection: with MIF (MIF) ELISA detection kit (on Hai Bairui bio tech ltd) by specification operation, detection range is 0～800pg/ml, and sensitivity is 1.0pg/ml, can Under white background, directly detecting by an unaided eye: in reacting hole, color is the deepest, the positive is the strongest, and negative reaction is colourless or the most shallow, depends on According to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: on ELISA detector, in 450nm (if with ABTS Colour developing, then 410nm) place, survey each hole OD value after returning to zero with blank control wells, if 2.1 times of the negative control OD value more than regulation, It is the positive, specifically presses the operation of test kit description.MIF integrates cytokine, somatomedin, hormone and enzyme characteristic Multi-effect protein molecular, plays the effect of central as the regulatory factor of inherent immunity and inflammatory reaction, in various infection and Active chronic inflammation disease plays panimmunity function.According to testing result, select the culture hole with stronger phagocytic function In cell clone repeat next round dilution cultivate, repeat 2-3 wheel, detection function-stable after take out, proceed to culture bottle expand Increase and cultivate.

4, the frozen and recovery of macrophage strain

Preserve first 12 hours and adjust cell growth state, take one bottle of growth vigorous, after the cell that form is good, suitably digestion Making cell suspension, 1000r/min is centrifuged 5min, removes supernatant, flicks and makes cell loose at the bottom of pipe, adds 4 DEG C of 9 parts preserved complete Full culture fluid and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, and cryopreservation tube is put into liquid nitrogen after taking-up by-70 DEG C of refrigerator overnight Container saves backup.Get out the hot water of about 40 DEG C before recovery, cryopreservation tube is carefully taken out from liquid nitrogen, be immediately placed in heat In water, uniformly shake makes cell thawing, is centrifuged 5min at 1000r/min, opens and freeze in superclean bench under aseptic condition after defrosting Depositing pipe, the cell complete culture solution after thawing washed once, and is then centrifuged 5min at 1000r/min, supernatant discarded, in case Make amplification cultivation.

5, the amplification of macrophage strain

Moving in culture bottle after using complete culture solution the most resuspended above-mentioned cell precipitation, put 37 DEG C, 5%CO2 cultivates Case is cultivated.Repeatedly pass on amplification cultivation, until required macrophage strain quantity, every Secondary Culture 10 generation, detect huge biting carefully The function of born of the same parents' strain, sees whether stable.Continue in several bottles, carry out large-scale culture, save backup.

The technical scheme of the application, the primary cell used includes being directly separated and huge the biting carefully in stem cell induction source Born of the same parents, dendritic cell, neurogliocyte, CD4+T cell, mononuclear cell and by chemical coupling, crosslinking, affine absorption system The granule being coated with CD4 molecule become, and/or the granule similar to CD4+ cell function.The construction method of immortalized cell line Hybridoma technology is fused including conventional cell.Plasmid vector includes selecting SV40LT/pLXSN, hTERT/pLPCX, SV40LT/ PcDNA3.1 (-) and hTERT/pLXSNneo.The composition causing immortalization includes reverse transcription virus gene, SV40LT, hTERT, EB Virus, oncogene, cell growth stimulant and derivant and include CD 3-resisting monoclonal antibody, AntiCD3 McAb/CD28 double antibody At interior CD4+ cell surface antibodies.

Two, the preparation of depurator

1, the filling of cleanser

The macrophage strain present invention prepared, after cleaning with physiological saline solution, then is centrifuged 5min with 1000r/min (low speed is centrifuged in short-term), takes cell precipitation assembling acrylate etc high-biocompatibility material (with plasma separator material phase With) the cylindrical cleaning device made, to 4/5, then add cells frozen storing liquid (containing 30% hyclone, 12% dimethyl sulfoxide RPMI-1640), make cell concentration reach 80%, shake up gently, sealing, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter- 196 DEG C of liquid nitrogen cryopreservations are standby.When defrosting uses, need be put in the 37 DEG C of water-baths having been warmed up by cryopreservation tube rapidly, and constantly Shake, makes the liquid in pipe melt rapidly, uses after then cleaning with physiological saline solution.When preparing cleanser, preserve thin The medium of born of the same parents includes selecting normal saline, regular growth culture fluid, cells frozen storing liquid.Macrophage strain preparation with phagocytosis HIV During cleanser, its concentration is 80%, and when preparing cleanser with the immunocyte and immune particle adsorbing HIV, its concentration is 95% Above.Various kinds of cell can be hybridly prepared into cleanser.

2, the specification of depurator

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

3, the material of depurator

Blood purification selects the macromolecular material of acrylate etc, it is desirable to good biocompatibility, activates benefit hardly Body, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can pass through covalency, The methods such as grafting, polymerization improve the structure of material, the regulation inhomogeneities on surface, hydrophilic, minimizing to blood coagulation and oxidative stress Impact thus improve biocompatibility, reduce complication generation.Hydrophilic gel is added, by 2 methyl-prop at adsorber inner surface Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate membrane, by controlling wet-spinning procedure, can generate CA/PMB30, CA/PMB80 and CA/PMB30-80, have higher blood and cell compatibility.Some had anticoagulation Material be solidificated on the material of carrier or adsorber inner surface, can suppress blood coagulation, improve biocompatibility, also can reduce Heparin consumption, and likely realize no-rod tractor.It is attached to heparin covalent polyether sulfone surface, both maintain the mechanics of polyether sulfone Performance, can improve again the anticoagulation function of adsorber inner surface.Covalent immobilisation linoleic acid film on cellulose acetate film, or by covalency It is attached to polyacrylic linoleic acid and is grafted onto polysulfone membrane surface, can have more preferable histocompatibility and anticoagulant effect. Along with macromolecular material and the development of nanotechnology, the material close with Human vascular endothelial will go out in the near future Existing.

Three, the preparation of separator

(1) preparation of blood separator

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

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

3, the model of blood separator: the profile of blood separator is prepared as column construction (with acetate fiber or absorbent cotton For material make filter element), flat structure (making filter element for material with poly-vinegar non-woven fabrics)；Aperture be prepared as 150～250 μm, 50 ～150 μm, 15～40 μm, 8～15 μm, 5～8 μm, 3～5 μm, 1～2 models of μm.

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

(2) preparation of plasma separator

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

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

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

Four, the component of acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument

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

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

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

Five, the connecting path of acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument and using method

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

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

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

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

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (15) is connected venous blood Pipe, then opens blood pump (2), and blood flow is 100～150ml/min, such as Fig. 1, when arterial blood through arterial blood line pipe (1), When heparin and blood pump (2) enter blood separator (3), the large volume multinucleated giant cell formed because of HIV is delayed at In blood separator (3), mononuclear blood cell and blood plasma export (4), blood pump (6) and circulation line (7) through blood successively and flow into Plasma separator (8), the blood plasma of separation flows into now open depurator (11) through blood plasma pump (9) and blood vessel (10) successively, Blood plasma to be full of, about 10 minutes, begin paying out blood plasma, through export pipeline (13) flow out, synchronize to depurator (12) irrigate blood plasma, When blood plasma in depurator (11) has nearly flowed, starting again at perfusion blood plasma, now depurator (12) begins paying out blood plasma, and two The depurator (11) of individual parallel connection and (12) are alternately.As represented Fig. 2 institute of the internal structure of the blood separator (3) in Fig. 1 Show, the tube wall of the inner chamber (2) of blood separator (1) has a lot of micropore (3), multinucleated giant cell (4) micropore (3) can not be filtered and Be delayed at inner chamber (2), thus can be eliminated, can by micropore (3), the mononuclear blood cell (5) of small size and blood plasma enters Enter exocoel (6), then flow out through outlet (7), and then isolate hemocyte and blood plasma through the plasma separator (8) shown in Fig. 1.As Shown in Fig. 3 of the internal structure of the plasma separator (8) in expression Fig. 1, the tube wall of the inner chamber (2) of plasma separator (1) has A lot of micropores (3), it is impossible to export (8) by the mononuclear blood cell (4) of micropore (3) through the hemocyte with switchable valve and flow Go out, enter the hemocyte export pipeline (14) shown in Fig. 1；Blood plasma can be entered by the blood plasma of micropore (3) and chemical composition (5) thereof Separator exocoel (6), then enters depurator through the blood plasma pump (9) shown in blood plasma flow export (7), Fig. 1 and blood vessel (10).As Represent in Fig. 1 shown in Fig. 4 of depurator (11) and (12) internal structure, when the blood plasma containing HIV (3) enters depurator (1), The macrophage phagocytic body (4) no longer moved down is formed, by clearly after macrophage (2) phagocytosis that HIV therein (3) is cleaned in device Except purifying individual cells that blood plasma separates through the export pipeline (13) shown in Fig. 1 with plasma separator (8) at outlet after HIV Conflux through venous line (15) after converging in road (14) place.So remove HIV, purify blood, until the plasma circulation being previously set Amount (usually 9L), treatment just ends, and whole therapeutic process is by computer control, and can detect duty at any time, makes By convenient, automatization and safety.

Six, the checking of acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument effect

1, blood separator filters the checking of HIV cell effect

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

The present inventor, according to the basic skills of the present invention, has done following simple confirmatory experiment: take Disease Control and Prevention Center and infection The some parts of anticoagulated whole blood of acquired immune deficiency syndrome (AIDS) (AIDS) patient made a definite diagnosis that sick laboratory biological Sample Storehouse preserves, respectively peek part phase It is mixed into 5 examples with the anticoagulated whole blood of abo blood group, makes blood flow volume sufficiently large, then entrust blood station, center to press the blood of composition blood transfusion Liquid becomes component separation, isolates leukocyte, erythrocyte, blood plasma through blood component piece-rate system, takes leukocyte composition routinely Centrifugation, inhales and abandons supernatant, and with appropriate normal saline suspension leukocyte cell pellet, the gp120 being subsequently adding proper ratio resists Body (Guang Rui bio tech ltd, Shanghai), mixes rearmounted 37 DEG C and reacts 5 minutes, then with blood that aperture is 20～30um Composition piece-rate system isolates the leukocyte (the biggest leukocyte) of large volume, to the leukocyte filtrate filtered further with hole Footpath be 15～25um blood component piece-rate system isolate the leukocyte (referred to as in leukocyte) of medium volume, white in filtrate Cell is the leukocyte (referred to as SL) of small size, collects large, medium and small leukocyte separation suspension respectively, and conventional centrifugal sinks Form sediment, inhale and abandon supernatant, with the large, medium and small leukocyte cell pellet of quantitative liquid shifter draws equal amounts respectively, conventional method (machinery or thin Cellular lysate liquid) cell lysis (as used lysate of the same race, need dosage equal), take supernatant after centrifugation, then 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 patient is not HIV-p24 content in the leukocyte of same volume size is different, and in large, medium and small leukocyte, the average content of HIV-p24 is respectively For 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, HIV-p24 average content difference in the most large and small leukocyte 148.6pg/ml, decrease 54.4%；In large, medium and small leukocyte the total content of HIV-P24 be respectively 1375.0pg/ml, 979.9pg/ml, 632.1pg/ml, in the most large and small leukocyte, HIV-p24 total content difference 742.9pg/ml, decreases 54.3%, through statistical test, t=2.43, p ＜ 0.05.Large volume leukocyte in AIDS patient body is described or resists through gp120 The large volume leukocyte formed after body effect contains the HIV of high level, can be by implementing technical scheme quilt Separate and remove.

2, the checking of HIV effect removed by depurator

In order to verify that effect of HIV is removed in macrophage strain, the present invention devises easy method of testing: take sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draws by centrifugation macrophage strain that (1000r/min, 5min) precipitate respectively to 200mm Scale, then draws and is incubated after 100 DEG C dissolve at 39～41 DEG C of 0.9% standby agarose C1-4B, reaches about 10mm length and carves Degree, after putting blood sedimentation tube cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but will not stop the water of little molecule Pass through with the material of chemical analysis etc.The acquired immune deficiency syndrome (AIDS) (AIDS) that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse preserve is suffered from 5 example blood plasma, the most about 10mL of person, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube (simple purifier) upper end sky in batches Pipe, wait flowing through the macrophage strain layer of blood sedimentation tube lower floor and after flowing out in blood sedimentation tube, collects effluent, blood after referred to as AIDS filter Slurry.Take blood plasma after the front blood plasma of AIDS filter and filter, with MIF (MIF) ELISA detection kit (Shanghai Hundred stamen bio tech ltd) pairing detection, by specification operates, and detection range is 0～800pg/ml, and sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, and the positive is the strongest, and negative reaction is nothing Color or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: on ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, surveys each hole OD value after returning to zero with blank control wells, if right more than the feminine gender of regulation According to 2.1 times of OD value, it is the positive.Result such as table 1, before filter, MIF testing result is feminine gender (or because content is less than inspection in blood plasma Survey sensitivity, blood plasma and preserve cause degraded etc. for a long time), and after filtering, in blood plasma, testing result is the positive.Illustrate that macrophage strain exists This process creates MIF cytokine.MIF is the multi-effect egg integrating cytokine, somatomedin, hormone and enzyme characteristic White molecule, as the effect of the regulatory factor performance central of inherent immunity and inflammatory reaction, scorching in various infection and acute and chronic Disease property disease plays panimmunity function.Filter before and after simple depurator while MIF pairing detection making AIDS blood plasma, this The pairing detection of HIV-1p24 has also been done in invention, and according to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), Shanghai is inspired raw Thing Science and Technology Ltd.) operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, The p24 antigen of 40pg/ml, 80pg/ml is less than 5pg/ml, measurement range 0～400pg/ml, line as comparison, lowest detectable limit Property scope 0.5pg/ml～80pg/ml, in 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is the highest In 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, the quilt as absorbance ＞ 0.12 Being considered positive, after result (table 2) illustrates that AIDS blood plasma filters simple purifier, part HIV is swallowed by macrophage strain Absorption, the blood plasma HIV after filtration significantly reduces, and after the 1st time filters, HIV clearance rate is 20.55%, after the 2nd time filters, HIV clearance rate is 42.83%, and p ＜ 0.01 has a significant effect, and illustrates that the increase HIV along with filtering number of times can be by constantly Remove, thus reach to treat AIDS purpose.

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

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

In a word, above-mentioned simple confirmatory experiment shows, is easily fused into large volume by the peripheral blood leucocyte of HIV many Core giant cell or many cells polymer, can be separated by blood separator and remove；And HIV free in blood plasma, can be by plasma purification Agent (macrophage strain) is removed, and shows that with separator and depurator (agent) be the acquired immune deficiency syndrome (AIDS) biological cell immunity that critical component is constituted Therapeutic instrument has the therapeutic efficiency significantly removing the inside and outside inhibition of HIV of hemocyte.

Claims

1. the acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument for medical domain, it is characterised in that by external made susceptible HIV Cell be formulated in, with certain concentration, the medium that favourable cell preserves, the exit made of perfusion high-biocompatibility material is arranged The depurator of screen cloth, makes depurator and made blood and plasma separator constitute the main part of extracorporeal circulation apparatus, is used for filtering and contains There is the HIV in the multinucleated giant cell of HIV and blood plasma.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 1, it is characterised in that described susceptible HIV's is thin Born of the same parents include being directly separated and/or the macrophage in stem cell induction source, dendritic cell, neurogliocyte, CD4+T are thin Born of the same parents, mononuclear cell and the granule being coated with CD4 molecule made by chemical coupling, crosslinking, affine absorption, and/or and CD4 The granule that+cell function is similar.

3. according to the arbitrary described acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument of claim 1 and 2, it is characterised in that described external The cell phalangeal cell of made susceptible HIV can infinitely expand in co-culturing with specific composition.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 3, it is characterised in that described specific composition is energy Make the SV40LT of cellular immortalization, hTERT, Epstein-Barr virus, oncogene, cell growth stimulant and derivant and CD4+ cell Surface antibody.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 4, it is characterised in that described CD4+ cell table Face antibody includes CD 3-resisting monoclonal antibody, AntiCD3 McAb/CD28 double antibody.

6. according to the arbitrary described acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument of claim 1 and 4, it is characterised in that for easily Sense cell transduction SV40LT, hTERT gene of HIV and to cause the carrier cloning of immortalization be SV40LT/pLXSN, hTERT/ PLPCX, SV40LT/pcDNA3.1 (-) and hTERT/pLXSNneo.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 1, it is characterised in that described certain concentration refers to The concentration of immunocyte and immune particle for swallow the concentration of the macrophage strain of HIV be 80%, for adsorbing HIV reaches More than 95%.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 1, it is characterised in that Jie preserved for cell Matter is containing 30% hyclone, the RPMI-1640 of 12% dimethyl sulfoxide.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 1, it is characterised in that described blood separator Inner chamber is communicated with exocoel by the micropore on tube wall, and the aperture of described micropore is 1～250 μm, can by single blood cell but Can not be by two and above mutual bonding or the large volume cell of fusion.

Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument the most according to claim 9, it is characterised in that selection micropore size is 150～250 μm, 50～150 the separator of μm separate the fungus of especially big volume, spirillum and/or tumor cell；Select micropore hole Footpath is that 15～40 μm, 8～15 μm, 5～8 μm, 3～5 mononuclear phagocyte of separator separation HIV of μm, multinuclear are big and small Born of the same parents, many cells polymer, the particulate matter being adsorbed with HIV and/or the easy CD4+ cell by HIV.

11. acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument according to claim 1, it is characterised in that the appearance of described depurator Amassing is 200～300ml, imports and exports and is equipped with cell screen cloth, and footpath, import department top sieve number is 800 mesh, footpath screen cloth at the bottom of exit Mesh number is 2.0～5.0 mesh, and liquid outlet arranges the cell strainer that mesh number is 100 mesh, arranges between liquid entrance and screen cloth The relief area of accelerating system stable circulation.

12. claim 1-11 arbitrary described acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument answering in preparing extracorporeal circulation apparatus With, it is characterised in that described extracorporeal circulation apparatus include one end of arterial blood line pipe (1) through heparin and blood pump (2) with contain The blood separator (3) of waste liquid outlet (5) is connected, and blood separator (3) exports (4), blood pump (6), circulation line through blood (7) being connected with plasma separator (8), the plasma outlet port of plasma separator (8) is through blood plasma pump (9) and blood vessel (10) with two also The depurator (11) of connection is connected with depurator (12), and the export pipeline (13) of two depurators is thin with the blood of plasma separator (8) Born of the same parents' export pipeline (14) confluxes through venous line (15) after converging.