CN106110425A - AIDS plasma purification therapeutic instrument - Google Patents

Abstract

nullA kind of AIDS plasma purification therapeutic 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，Preparation had not only retained phagocytosis characteristic but also can the hybridoma macrophage strain of indeterminate growth，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 cleaned agent and removes，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

AIDS plasma purification therapeutic instrument

Technical field

The present invention relates to preparation and the application of AIDS plasma purification therapeutic instrument in medical domain, be mainly used in HIV sufferers The removing of the inside and outside HIV (human immunodeficiency virus) of hemocyte, thus reach to treat the purpose of acquired immune deficiency syndrome (AIDS).

Background technology

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 Being formed by the monocyte precursor Development And Differentiation in bone marrow, account for 3% one the 8% of blood middle leukocytes sum, its volume relatively drenches Bar cell is bigger, and mononuclear cell the most only stops 12-24 hour, subsequently into connective tissue or organ, reach maturity into Macrophage, macrophage is the differentiation of mononuclear phagocyte system camber, ripe cell type, has stronger phagocytosis merit Can, wandering macrophage is more than mononuclear cell several times, lasts a long time, some months of can surviving in the tissue, the macrophage settled down Having different titles, be Kupffer Cell, be microglia in brain, be osteoclast etc. in bone in liver, it expresses Fc Receptor, C3b receptor and CDl4, play defense function in inherent immunity, is also that the professional antigen participating in adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of HIV (human immunodeficiency virus) (HIV), after HIV enters human body, first suffers huge biting carefully The phagocytosis of born of the same parents, but HIV quickly changes the sour environment at macrophage some position interior, creates condition of its existence applicable, The most it is not killed amount reproduction the most within it to assemble, then HIV is passed to CD4+T cell.So, can be with conventional hybridization Oncocyte technology of preparing, prepares macrophage hybridoma, for preparing the depurator for the treatment of acquired immune deficiency syndrome (AIDS) after a large amount of amplifications, The HIV in blood plasma is removed with the phagocytic function of macrophage.

In a word, various medicines and biological product cannot effectively kill internal HIV (human immunodeficiency virus), and price, and side effect is big, So far there is no the effective ways for the treatment of acquired immune deficiency syndrome (AIDS), it has also become attack the global problem being unable to for a long time.

Summary of the invention

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

The invention aims to provide AIDS plasma purification therapeutic instrument；Another object is intended to provide AIDS plasma purification to control Treat preparation and the application process of instrument.

The object of the present invention is achieved like this: it is thin that preparation can filter the large volume containing HIV that many cells are combined into The blood separator of born of the same parents and can separate the plasma separator of mononuclear blood cell and blood plasma, had both retained former huge with hybridoma technology preparation Phagocyte characteristic can expand again parallel in the hybridoma macrophage strain of indeterminate growth, takes macrophage strain and is formulated in cell cryopreservation Liquid, makes cell concentration reach 80%, and what then perfusion high-biocompatibility material was made can prevent cell and fragment thereof from leaching and energy The plasma purification device in place is provided for cell strain phagocytosis HIV, during wherein macrophage strain is fixed on plasma purification device, plays phagocytosis HIV is used, made acquired immune deficiency syndrome (AIDS) plasma purification device so that with blood, plasma separator combination and additional computer regulatory process and Preparation AIDS plasma purification therapeutic instrument, the blood separator that the blood in extracorporeal circulation is treated in instrument filters the multinuclear of large volume Giant cell, and then it is divided into blood plasma and mononuclear blood cell by plasma separator, with mononuclear blood cell after blood plasma purified device removing HIV Converge feedback.

The present invention is made up of blood, plasma separator and plasma purification device, 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 removed by plasma purification device, the blood plasma after purification Feed back after converging with the mononuclear blood cell of medium volume, thus remove and be combined in cell surface and/or intracellular HIV and trip From in 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 Anti-reverse transcription drug treatment.

Detailed description of the invention

Fig. 1 is the application schematic diagram of the AIDS plasma purification therapeutic 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 embodiment of the AIDS plasma purification therapeutic instrument that the present invention proposes is made Detailed description.

One, the preparation of hybridoma macrophage strain

1, the source of primary cell

(1) single core blood cell: refer to the lymphocyte separated from blood with density－gradient centrifuga－tion method and monokaryon is huge bites Cell.Concrete grammar is: the White Blood Cells Concentrate buying Blood Center or the Cord blood preserved for scientific research, takes 2mL specimen, PBS 6mL anticoagulation dropper, by hemodilution 2～3 times, is fully slowly superimposed on along tube wall after mixing that to have added 4mL lymph thin by liquid Born of the same parents separate in the 10mL centrifuge tube of liquid horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；It is divided in pipe after Li Xin 3 layers, upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, in upper, middle level Interface has the white cloud and mist layer narrow band based on mononuclearcell to be PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC inserts in another 50mL centrifuge tube, adds 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new life Sanguis Bovis seu Bubali + 2mmol/LEDTA clearly, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add counted under microscope 4 on blood counting chamber Cell (PBMC) sum in individual block plaid.

(2) single core histiocyte: provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, its preparation method is: the 1. acquisition of spleen tissue and transhipment: in the approval of reason committee and patient's informed consent Under, take the spleen specimen tissue of excision, shred into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to rapidly cell culture chamber.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in-house blood and to ensure the aseptic of tissue.Machine Tool grinds spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 liquid.By 200 mesh stainless steel filtering net mistakes Filter is outstanding is mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing erythrocyte, lymphocyte, Macrophage etc.).3. erythrocytic cracking in spleen tissue cell suspension: then centrifugal with the washing of RPMI-1640 liquid (1000r/min, 3min), to remove cell debris, adds Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), removes the splitting erythrocyte fragment in supernatant, centrifugal 3 times of PBS washing, RPMI-1640 washing from The heart 1 time, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, now, mainly contains in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as cultivation Cell stock solution, Trypan Blue judges vigor and counts, and adjusting cell concentration with RPMI-1640 liquid is (3～5) × 10⁶/ L, will Adjusting the cell suspension inoculation of concentration in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2～3h, under phase contrast microscope observe form.The digestion of adherent spleen tissue macrophage: adherent spleen tissue is huge to be bitten The digestion of cell: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, and the washing of gained cell suspension is centrifugal (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or the discarded specimen of Post operation carries Take preparation, such as peritoneal cavity liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa etc..

(3) amniotic fluid, villus cell: attached hospital for obstetrics and gynaecology of Zhejiang University reproduction genetic laboratory is standby.Reason committee member Can ratify with under patient's informed consent, treating excess syndrome tests the residue amniotic fluid after diagnosis report, villus cell, selects exponential phase cell Continue to employ.

2, cell is cultivated and the adherent preliminary sorting of macrophage

Cell is cultivated routinely, but according to the difference of cellularity, suitably adjusts incubation time, condition of culture etc., typically Single core blood cell (PBMC) or single core histiocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method Culture dish in, in 37 DEG C, 5%CO₂Cell culture incubator (Themo electro corporation CLASS 100, beautiful State) in hatch 2h, after mononuclearcell is adherent, inhale abandon upper strata suspension cell (cell beyond macrophage be difficult to adherent and Remove with upper liquid), PBs buffer washs 3 times gently, adds a small amount of RPMI 1 culture medium, scrapes patch with cell scraper Parietal cell (predominantly macrophage, but also have other attached cells a small amount of).1000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid is thin Born of the same parents, villus cell are cultivated 1～7 day, occur that cell growth clone, cell growth converge rate and reach the exponential phase of 60～80% Cell, with trypsinization, PBS, obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorting

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

4, CDl4 cell (macrophage) sorting

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

Basic skills is analogous to cd4 cell, uses immunomagnetic beads method.1. reagent: people's CDl4 immunomagnetic beads test kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead is with special Opposite sex target cell one mononuclear cell combines: every 1 × 10⁸Individual PBMC adds magnetic bead and the 800uL buffering of 200uL coupling CDl4 antibody Liquid (containing the bovine serum albumin 2.5mL and 2mol/L EDTA0.5mL of 10%, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube Fully mixing, hatches 15min for 4 DEG C, and centre can slightly shake 1 time.Taking-up centrifuge tube after 15min, every 1 × 10⁷Individual cell adds 1 ～2mL pre-cooling buffer, 1000r/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.1000r/min is centrifuged 5min, abandons supernatant.2. flow cytometry Method: CDl4 labelling: take PBMC, adjusts with buffer (bovine serum albumin 2.5mL and 2mol/LEDTA 0.5mL containing 10%) Whole cell density is 1 × 10⁸/ mL, adds CDl4+-FITC antibody 100uL, 4 DEG C of lucifuge labellings in every milliliter of cell suspension 18min, then addition 1mL streaming buffer is to terminate dyeing in centrifuge tube, PBs washs 3 times, with the PBS containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation at flow cytometer (BD FAcsAria II, U.S.) upper sorting, according to the fluorescence intensity of CDl4 antibody, the relative size of cell and the relative particle of cell and interior The complexity of portion's structure, collects the cell of CDl4+.

5, prepared by CDl4 hybridoma cell strain (hybridoma macrophage strain)

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire cattle Jinshi City Hao on daytime ocean biological product science and technology responsibility company limited purchased by serum (FBS)；DMSO (--methyl sulfoxide) it is domestic analytical pure Reagent.

(2) myeloma cell prepares: merges and takes out the myeloma cell (SP2/ that a pipe is frozen the last week in liquid nitrogen container 0), it is immediately placed in hot water and thaws that (applying most is Sp2/0 cell strain, and this cell strain growth and fusion efficiencies are the best, and itself is not Secreting any heavy chain immunoglobulin or light chain, the Seedling height scale of cell is 9 × 10⁵/ ml, the doubling time be usually 10～ 15h；The actual application relevant to human body considers select homologous cell strain, if Fu Xiang bio tech ltd, Shanghai is to ATCC The NCI-H929 human myeloma cell strain that cell bank is introduced).Adding appropriate complete culture solution after thawing, 1000r/m is centrifuged 3min； It is repeated 1 times.Precipitate is moved in Tissue Culture Flask, add DMEM culture fluid, put CO2 incubator and cultivate, within 3-4 days, once pass Generation or amplification culture, adjust cell state in merging first 24 hours, it is ensured that before merging, cellular morphology is good, it is vigorous to grow.Add Appropriate basal medium is in centrifuge tube, and after beaing mixing gently, 1000r/m is centrifuged 5-10min, repeated washing cell 2 times.

(3) CDl4 cell (macrophage) to be hybridized prepares: the mononuclear phagocyte of present invention sorting is with basal medium Adjust total cell and count to 1 × 10⁸～2 × 10⁸Merge for cell.Blue dyeing phase-contrast microscopy, viable count is expected with platform Should be higher than that 80% for qualified.

(4) cell merges: by CDl4 cell (mononuclear phagocyte) with myeloma cell with 10: the ratio of 1-5: 1 adds In centrifuge tube, being mixed evenly, 1000r/m is centrifuged 5min, supernatant discarded, beats gently at the bottom of pipe to cell without granular precipitate, weight Multiple 2 times.Preheating centrifuge tube is rotated gently, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up in 37 DEG C of water-baths PEG3000 is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, afterwards by the basis training of the 25mL of preheating Support base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, rotating centrifugal pipe lightly during adding, then stand In 37 DEG C of water-bath 10min, 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T culture medium.Suitably it is inoculated into after mixing In 96 well culture plates, it is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.

(5) there is the mononuclear phagocyte strain screening of phagocytic function: observe cell growth status in 96 well culture plates, 7-10 Only have hybridoma after it and can grow division, now discard HAT culture medium, change complete medium.Cell clone grows When area reaches 1/10 cell hole, culture supernatant, selection is gone to have the culture hole of the hybridoma cell strain that growth conditions is good, aobvious The position of labelling cell strain growth, size under micro mirror, using aseptic rifle head to draw cell clone in the position of mark has to new In the culture hole of complete medium, doubling dilution counts hole to below the most successively, 37 DEG C, cultivates one week left side in 5%CO2 incubator The right side, basis of microscopic observation cell growth status, when cell clone covers with to hole floor space more than 1/10, take in cell or cultivation Clear detection hybridoma macrophage (M φ) strain function.

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

2. hybridoma macrophage strain phagocytosis HIV Function detection: take acquired immune deficiency syndrome (AIDS) (AIDS) patient's of Disease Control and Prevention Center's preservation After blood plasma is mixed with hybridoma macrophage strain, separates cell strain, PBS 3 times, measure the phagocyte strain through cracking The function of phagocytosis HIV, with specific reference to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), Shanghai inspires biotechnology limited Company) operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is less than 5pg/ml, measurement range 0～400pg/ml, the range of linearity as comparison, lowest detectable limit In 0.5pg/ml～80pg/ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, is recognized as absorbance ＞ 0.12 For being the positive.Concrete test kit description of pressing operates.

3. the strain of hybridoma macrophage produces macrophage cytokines detection: with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) by specification operates, and detection range is 0～800pg/ml, Sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, and the positive is the strongest, negative Reaction for colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: at ELISA detector On, in 450nm (if developing the color with ABTS, then 410nm) place, survey each hole OD value after returning to zero with blank control wells, if more than regulation 2.1 times of negative control OD value, are the positive, specifically press the operation of test kit description.MIF be collection cytokine, somatomedin, Hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays central Effect, in various infection and active chronic inflammation disease play panimmunity function.

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

(6) preservation of hybridoma macrophage strain and recovery: preserve first 12 hours and adjust cell growth state, take one bottle of life Making cell suspension after long vigorous, that form is good cell, suitably digestion, 1000r/min is centrifuged 5min, removes supernatant, flicks Making cell loose at the bottom of pipe, add 4 DEG C of 9 parts of complete culture solutions preserved and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, and-70 DEG C refrigerator overnight, puts into cryopreservation tube in liquid nitrogen container after taking-up and saves backup.The hot water of about 40 DEG C is got out before recovery, will Cryopreservation tube carefully takes out from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, is centrifuged at 1000r/min after defrosting 5min, opens cryopreservation tube under aseptic condition in superclean bench, and the cell complete culture solution after thawing washed once, then It is centrifuged 5min, supernatant discarded, in case making amplification culture at 1000r/min.

6, prepared by hybridoma macrophage strain treatment cell

The i.e. amplification cultivation of hybridoma macrophage strain.Move after using complete culture solution the most resuspended above-mentioned cell precipitation Enter in culture bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Repeatedly pass on amplification cultivation, until required hybridoma cell strain Quantity, every Secondary Culture positive hybridoma cell strain 10 generation, the function of detection macrophage hybridoma cell strain, see whether steady Fixed.Continue in several bottles, carry out extensive industrialization to prepare, save backup.

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

1, the filling of cleanser

The hybridoma macrophage strain present invention prepared, after cleaning with physiological saline solution, then is centrifuged with 1000r/min 5min (low speed is centrifuged in short-term), takes cell precipitation assembling acrylate etc high-biocompatibility material (with plasma separator material Expect identical) 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 standby.When defrosting uses, need put in the 37 DEG C of water-baths having been warmed up by cryopreservation tube, not rapidly Disconnected shake, makes the liquid in pipe melt rapidly, uses after then cleaning with physiological saline solution.

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 AIDS plasma purification therapeutic 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 AIDS plasma purification therapeutic 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 of the internal structure of the blood separator (3) in Fig. 1, blood There are a lot of micropore (3), multinucleated giant cell (4) micropore (3) can not be filtered and hindered on the tube wall of the inner chamber (2) of liquid/gas separator (1) Stay inner chamber (2), thus can be eliminated, can pass through in micropore (3), outside the mononuclear blood cell (5) of small size and blood plasma enters Chamber (6), then flows out through outlet (7), and then isolates hemocyte and blood plasma through the plasma separator (8) shown in Fig. 1.As represented Fig. 3 of the internal structure of the plasma separator (8) in Fig. 1, the tube wall of the inner chamber (2) of plasma separator (1) has a lot of micropore (3), it is impossible to export (8) by the mononuclear blood cell (4) of micropore (3) through the hemocyte with switchable valve and flow out, enter Fig. 1 Shown hemocyte export pipeline (14)；Plasma separator exocoel can be entered by the blood plasma of micropore (3) and chemical composition (5) thereof (6), then depurator is entered through the blood plasma pump (9) shown in blood plasma flow export (7), Fig. 1 and blood vessel (10).As represented in Fig. 1 Depurator (11) and Fig. 4 of (12) internal structure, when the blood plasma containing HIV (3) enters depurator (1), HIV therein (3) Forming, after macrophage (2) phagocytosis being cleaned in device, the macrophage phagocytic body (4) no longer moved down, be eliminated after HIV is clean The individual cells that change blood plasma separates with plasma separator (8) through the export pipeline (13) shown in Fig. 1 converges at export pipeline (14) Conflux by venous line (15).So remove HIV, purify blood, until the plasma circulation amount being previously set (usually 9L), Treatment just ends, and whole therapeutic process is by computer control, and can detect duty, easy to use, automatization at any time And safety.

Six, the checking of AIDS plasma purification therapeutic instrument effect

1, blood separator filters the checking of HIV cell effect

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

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

2, the checking of HIV effect removed by depurator

In order to effect of HIV is removed in check cross tumor macrophage strain, the present invention devises easy method of testing: takes and goes out 2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5, draws by centrifugation the macrophage hybridization that (1000r/min, 5min) precipitates respectively Tumor cell strain, to 200mm scale, is then drawn and is incubated after 100 DEG C dissolve at 39～41 DEG C of 0.9% standby agarose C1- 4B, reaches the long scale of about 10mm, and after putting blood sedimentation tube cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but The material of water and the chemical analysis that will not stop little molecule etc passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse are protected 5 example blood plasma, the most about 10mL of acquired immune deficiency syndrome (AIDS) (AIDS) patient deposited, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube (letter in batches Easily purifier) upper end blank pipe, wait flowing through the hybridoma macrophage strain layer of blood sedimentation tube lower floor and after flowing out in blood sedimentation tube, receive Collection effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) pairing detection, by specification operates, detection range Being 0～800pg/ml, sensitivity is 1.0pg/ml, directly can detect by an unaided eye under white background: in reacting hole, color is more Deeply, the positive is the strongest, and negative reaction is colourless or the most shallow, according to the depth in color, with "+", "-number represent.Also OD can be surveyed Value: on ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, surveys each hole OD after returning to zero with blank control wells Value, if 2.1 times of the negative control OD value more than regulation, it is the positive.Result such as table 1, before filter, in blood plasma, MIF testing result is equal For negative (or because content preserves cause degraded etc. for a long time less than detection sensitivity, blood plasma), and after filtering, in blood plasma, testing result is Positive.Illustrate that macrophage hybridoma cell strain creates MIF cytokine in this process.MIF be collection cytokine, growth because of Son, hormone and enzyme characteristic multi-effect protein molecular, as in the regulatory factor performance of inherent immunity and inflammatory reaction The effect of pivot, plays panimmunity function in various infection and active chronic inflammation disease.Letter is filtered making AIDS blood plasma Easily before and after depurator while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 antigen Detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operates, with concentration known 0pg/ml, 0.5pg/ The p24 antigen of ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, lowest detectable limit Less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, in 15min, 450nm measures extinction Degree (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ml extinction Degree is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and result (table 2) illustrates that AIDS blood plasma filters simple purification After device, part HIV is by the phagocytosis absorption of macrophage hybridoma cell strain, and the blood plasma HIV after filtration significantly reduces, through the 1st After secondary filtration, HIV clearance rate is 20.55%, and after the 2nd time filters, HIV clearance rate is 42.83%, p ＜ 0.01, has substantially Effect, illustrate along with filter number of times increase HIV can constantly be removed, thus reach treat AIDS purpose.

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

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

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

Claims (10)

1. the AIDS plasma purification therapeutic instrument for medical domain, it is characterised in that by the made hybridoma that can swallow HIV Macrophage strain is formulated in cells frozen storing liquid, and the exit that perfusion high-biocompatibility material is made arranges the depurator of screen cloth, Making macrophage strain concentration reach 80%, made depurator constitutes the master of purging in vitro device with made blood and plasma separator Part, for filtering the HIV in the multinucleated giant cell containing HIV and blood plasma.

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

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

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

AIDS plasma purification therapeutic instrument the most according to claim 1, it is characterised in that described plasma separator hollow fibre Film a diameter of 270～370 μm, film thickness is 50 μm, and aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm, can stop All of cell filters.

AIDS plasma purification therapeutic instrument the most according to claim 1, it is characterised in that described depurator is by its exit Screen cloth and hybridoma macrophage strain collectively form molecular sieve mechanical removal and the barrier of cellular immunization removing blood plasma HIV.

AIDS plasma purification therapeutic instrument the most according to claim 6, it is characterised in that the volume of described depurator is 200 ～300ml, to import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh, and at the bottom of exit, footpath sieve number is 2.0～5.0 mesh, liquid outlet arranges the cell strainer that mesh number is 100 mesh, arranges promotion system between liquid entrance and screen cloth The relief area of system stable circulation.

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

9. the preparation for the AIDS plasma purification therapeutic instrument cleanser of medical domain, it is characterised in that hybridoma is huge Phagocyte strain is cleaned with physiological saline solution, takes the circle that sedimentation cell assembling acrylate etc high-biocompatibility material is made Cylindricality depurator, to 4/5, then adds cells frozen storing liquid, makes cell concentration reach 80%, shake up gently, sealing, through 4 DEG C, and 0.5h；- 20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen cryopreservations standby, when defrosting uses, need to rapidly cryopreservation tube be put into the most pre- In 37 DEG C of water-baths of heat, and constantly shake, make the liquid in pipe melt rapidly, use after then cleaning with physiological saline solution.

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