CN106178162B - Treating AIDS organelle - Google Patents

Abstract

The present invention relates to the treating AIDS organelles of field of biomedicine, it is characterized in that isolating CD14 cell with immunomagnetic beads method, and then it prepares and had not only retained former macrophage feature but also the hybridoma macrophage strain of energy indeterminate growth, it selects to make amplification cultivation and preservation to the strong phagocytic clone of HIV, thus the hybridoma macrophage strain prepared prepared with high-biocompatibility material package can prevent cell and its fragment even HIV from filtering out, HIV capable of being swallowed for cell strain, the organelle in place is provided, extracorporal circulatory system is constituted with plasma separator, isolated blood plasma is after organelle filters out, HIV therein is swallowed by macrophage strain to be removed, the anti-infective macrophage cytokines generated simultaneously feed back internal with blood plasma, the AIDS hybridoma technology of macrophage cytokines needed for not only having removed HIV to realization but also supplemented is external New treatment, than being confined to kill HIV in vivo but the traditional remedies that are difficult to realize are more feasible and have no toxic side effect.

Description

Treating AIDS organelle

Technical field

The present invention relates to treating AIDS organelles in field of biomedicine, and it is sensitive mainly to prepare HIV by artificial industry Cell achievees the purpose that treat AIDS for swallowing the free HIV in blood plasma in vitro.

Background technique

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

HIV is the retrovirus for infecting human immune cells, and about 120 nanometers of diameter, substantially spherical in shape, outer membrane is lipoid Coating (comes from host cell), and embedding virulent albumen gp120 and gp41, gp41 are transmembrane proteins, and gp120 is located at surface, And with gp41 by noncovalent interaction in conjunction with, be inward from by the albumen p17 sphere matrix (Matrix) formed and albumen p24 The half-cone capsid (Capsid) of formation, capsid include virulent rna gene group, enzyme (reverse transcriptase, integrase, protease) And other ingredients from host cell.

After HIV enters human body, first by the phagocytosis of macrophage, but HIV changes certain portions in macrophage quickly The acidic environment of position creates the condition for being suitble to it to survive, and is not killed mass propagation aggregation in it instead not only.Because CD4 is the receptor of HIV, so the HIV bred in macrophage is by its envelope protein gp120 and under the auxiliary of gp41 It is (thin that (gp41 plays a part of bridge, utilizes itself hydrophobic effect mediate retroviral cyst membrane and cell membrane fusion) enters CD4+ cell Born of the same parents, mononuclear macrophage, Dendritic Cells etc.), it is proliferated rapidly in the cell, generates 10 daily⁹~10¹⁰Virion, not Disconnected other normal and regenerated CD4+ that enter are replicated into the cell, are manufactured more virus infected cells, are kept peripheral blood CD4+T thin Born of the same parents' sustaining breakdown is reduced.CD4+T cell is most important immunocyte, and the infected once loses a large amount of CD4+T cells, whole A immune system will all lose resistance to the infection of various diseases by deathblow；HIV enters host's CD4+ cell It can also show as hiding without showing clinical symptoms for a long time afterwards, geneome RNA reverse transcription is whole with virus at double-stranded DNA Synthase enters in host cell nuclear, and under the action of integrase, double-stranded DNA is integrated into host cell gene group, is integrated Viral DNA is known as provirus, and the several months that can hide does not replicate even for many years, causes the incubation period of AIDS several months to many years.In AIDS Incubation period, HIV mainly breeds in the macrophage of lymph node and Dendritic Cells, these cells are intracorporal HIV storages Library, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, mitogen, antigen, TNF, IL-2 and lymph element (LT) HIV provirus gene can be excited to replicate in the CD4+T Intracellular transcription of infection.After being largely proliferated, inhibition of HIV particle Blood is constantly discharged and be free on from the infection cell being destroyed, and is then entered back into other cells and is continued course of infection.

Body production envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody can be stimulated after HIV infection.? Low-level antiviral neutralizing antibody is measured in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers' highest illustrates that the antibody has protective effect in vivo.But antibody cannot be with the virus that retains in mononuclear macrophage Contact, and antigenic variation easily occurs for HIV envelope protein, original antibody is ineffective, keeps neutralizing antibody due from playing Effect.In the latent infection stage, HIV provirus is integrated into host cell gene group, therefore HIV will not be known by immune system Not, so relying solely on itself immune function can not be removed.Another critically important reason is to be killed, removed according to antibody After the mechanism of antigen, immune antibody and antigen binding, to generate immunological effect or by activating complement, mediate ADCC effect Cellular antigen should be dissolved, but HIV is not cellular antigen；Phagocyte phagocytosis is attracted to remove by chemotaxis anti- Original, but HIV is protected in phagocyte instead, is proliferated；Antibody and antigen binding play neutralization, make to lose infection Power, but HIV can not be killed by immune system, be removed.

The blood purification technology that 20th century began one's study refers to outside the blood lead body patient and is filled by a kind of purification Set, remove some of them morbid substance, purify blood, then feed back into vivo, achieve the purpose that treat disease.The blood of early stage is net Change and often refer to hemodialysis technology, uremic patient kidney replacement therapy is used for, with the progress of medicine and the hair of apparatus for purifying blood Exhibition, derives different blood purification modes, such as plasma exchange, blood perfusion, immuno absorbence, blood on the basis of haemodialysis Liquid filtration etc..The application of different blood purification technologies substantially improves the treatment of certain difficult complex diseases, mainly applies blood plasma Replacement technique separates and removes macromolecular immune complex class virulence factor to mitigate, adjust and treat certain immunity diseases. Since the 1980s, the First Academy of Zhejiang University Li Lan Juan team starts to achieve good therapeutic effect with replacement therapy of blood plasma hepatic failure, And abiotic type, bion, hybrid artificial liver support system and the dialysis of related artificial liver have been founded on this basis The improvement and innovation of the artificial liver system unit such as device, transfusion filters, active carbon (resin) absorber, bioreactor. But it has no and HIV infection cell and the report of free HIV is removed with blood purification technology.

Commonly antiviral first-line drug is reverse transcriptase inhibitor to treating AIDS at present, which cannot kill HIV, deposit Individual difference, drug resistance, gland plastochondria toxicity, bone marrow suppression, globular anemia, granulocyte and decrease of platelet, pancreatitis, A variety of toxic side effects such as hepatic injury, other as hiv protease inhibitor, hiv integrase inhibitor, cell factor, vaccine therapy, Gene therapy and monoclonal antibody passive immunization therapy etc. are difficult to because of a variety of causes commonly used.There is document report sharp The stimulant for using the monoclonal antibody of T cell surface C D3 molecule to grow as cell, the T of mass propgation AIDS patient separation It after cell, is fed back as itself therapeutic cells, but HIV is bred also with the culture of HIV infection cell in endogenous multiplication, is increased The feedback of amount T cell also results in the feedback of increment HIV.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophil leucocyte in peripheral blood, macrophagocyte It is the macrophage in the monocyte and a variety of organs, tissue in blood, the two constitutes mononuclear phagocyte system.Monocyte It is formed by the monocyte precursor Development And Differentiation in marrow, accounts for about 3% one the 8% of blood middle leukocytes sum, volume is relatively drenched Bar cell is bigger, and monocyte only stops 12-24 hours in blood, subsequently into connective tissue or organ, reach maturity for Macrophage, macrophage are highly differentiation, mature cell type in mononuclear phagocyte system, have stronger phagocytosis function Can, wandering macrophage is greater than monocyte several times, and it lasts a long time, can survive in the tissue some months, the macrophage of colonization There is different titles, be Kupffer Cell in liver, be in brain microglia, be osteoclast etc. in bone, expresses Fc Receptor, C3b receptor and CDl4 play defense function in inherent immunity, and the professional antigen of participation adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of AIDS virus (HIV), thin by macrophage first after HIV enters human body The phagocytosis of born of the same parents, but HIV changes the acidic environment at certain positions in macrophage quickly, creates the condition for being suitble to it to survive, It is not killed mass propagation aggregation in it instead not only, then HIV is passed into CD4+T cell.

In conclusion the CD4 molecule of Expression of Macrophages, is the receptor of AIDS virus (HIV), it is first after HIV enters human body First by the phagocytosis of macrophage, but HIV changes the acidic environment at certain positions in macrophage quickly, creates and is suitble to it The condition of existence is not killed mass propagation aggregation in it instead not only, then HIV is passed to CD4+T cell, last dead In the various diseases that CD4+T cell considerable damage, function are lost and cause.

Human body lacks the immunity for killing HIV, and the curative effect of medication used in vivo is bad and toxic side effect is big, and AIDS is long Attack the global problem being unable to.

Summary of the invention

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

The invention aims to provide treating AIDS organelle；Another object is to provide for the preparation of organelle and answers Use method.

The object of the present invention is achieved like this: isolating CDl4 cell with immunomagnetic beads method, and then prepares and both retain original The hybridoma macrophage strain of macrophage feature and energy indeterminate growth selects to make amplification cultivation to the strong phagocytic clone of HIV And preservation, by the hybridoma macrophage strain thus prepared with high-biocompatibility material package and prepare can prevent cell and its Fragment even HIV filter out, can be cell strain phagocytosis HIV the organelle in place be provided, with plasma separator composition extracorporal circulatory system, Isolated blood plasma is after organelle filters out, and HIV therein is swallowed by macrophage strain to be removed, while the anti-infective macrophage generated is thin Intracellular cytokine is fed back in vivo with blood plasma, to realize the AIDS hybridoma skill not only removed HIV but also supplemented required macrophage cytokines The external new treatment of art, than being confined to kill HIV in vivo but the traditional remedies that are difficult to realize are more feasible and have no toxic side effect.

The present invention separates macrophage with classical magnetic activated cell seperation, thin with classical hybridoma technology preparation macrophage Born of the same parents' hybridoma cell strain is allowed to not only retain the activity of phagocytosis HIV, but also myeloma cell's characteristic with indeterminate growth, through expanding Treating AIDS organelle is made with high-biocompatibility material package afterwards, the CD4 molecule of Expression of Macrophages therein is Chinese mugwort The receptor of virus to be grown, there is the characteristic of natural phagocytosis HIV, the present invention simulates non-specific phagocytosis mode in macrophage body, when When blood plasma containing HIV flows through organelle, HIV is in contact with macrophage and is swallowed, and the blood plasma and blood after removing HIV are thin Born of the same parents feed back after converging, and internal HIV can be made constantly to be reduced by removing in the treatment until disappearing, can effectively block newborn CD4+ T cell is again by HIV infection, to reach the therapeutic purposes of immunologic reconstitution, and is difficult to kill HIV in vivo at present and be difficult to effective It blocks again to the infection cycle approach of CD4+T cell from blood plasma to macrophage, and somewhat expensive, the big routine of medicine side effect are anti- Reverse transcription treatment is compared, and the present invention is more economical, almost without medicine side effect, realize manually by HIV from be transferred in vivo it is external and The treatment new method of removing.

Specific embodiment

Fig. 1 is the application schematic diagram of the treating AIDS organelle proposed according to the present invention.

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

Fig. 3 is the internal structure chart of the treating AIDS organelle proposed according to the present invention.

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

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

In Fig. 3,1 is organelle, and 2 be hybridoma macrophage strain, and 3 be the free HIV into organelle, 4 be HIV with The conjugate of hybridoma macrophage strain, 5 be the cell factor that hybridoma macrophage strain generates.

Below with reference to Fig. 1, Fig. 2 and Fig. 3, the embodiment for the treatment of AIDS organelle proposed by the present invention is made detailed Description.

1, primary cell source

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

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

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

2, cell culture and the adherent preliminary sorting of macrophage

Routinely cell culture, but according to the difference of cellularity, appropriate adjustment incubation time, condition of culture etc., generally Single core blood cell (PBMC) or single core histocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method Culture dish in, in 37 DEG C, 5%CO₂Cell incubator (Themo electro corporation CLASS 100, beauty State) in be incubated for 2h, after mononuclearcell is adherent, inhale abandon upper layer suspension cell (cell other than macrophage be not easy it is adherent and Removed with upper liquid), PBs buffer gently washs 3 times, and a small amount of mono- 1640 culture medium of RPMI is added, scrapes patch with cell scraper Parietal cell (predominantly macrophage, but there are also other a small amount of attached cells).1000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid is thin There is cell growth clone in born of the same parents, villus cell culture 1~7 day, cell growth converges the logarithmic growth phase that rate reaches 60~80% Cell is digested with pancreatin, and PBS cleaning obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorts

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

4, CDl4 cell (macrophage) sorts

CDl4 is monocyte and the distinctive surface marker of macrophage, theoretically if from single core histocyte, sheep It is sorted in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, gained Cell includes monocyte and macrophage；But because the monocyte service life is short, only survive 1 day in peripheral blood and can not show a candle to macrophage Cell is easy to adherent growth, so removing substantially in cell adhere-wall culture of the invention, the cell sorted out is essentially Macrophage.

Basic skills is analogous to cd4 cell, using immunomagnetic beads method.1. reagent: people's CDl4 immunomagnetic beads kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead and spy Anisotropic one monocyte of target cell combines: every 1 × 10⁸The magnetic bead and 800uL buffering of 200uL coupling CDl4 antibody is added in a PBMC Liquid (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/L EDTA0.5mL, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube It mixes well, 4 DEG C of incubation 15min, centre can slightly shake 1 time.Take out centrifuge tube after 15min, every 1 × 10⁷A cell is added 1 Buffer is pre-chilled in~2mL, and 1000r/min is centrifuged 8min, abandons supernatant, and 0.5mL buffer is added and blows and beats into single cell suspension. (B) it collects the monocyte of marked by magnetic bead: cell splitter is placed on MACS magnetic frame, 1mL buffer statocyte is added Splitter drips to no liquid, immediately adds above-mentioned cell suspension in people's cell splitter, rinses cell with 0.5mL buffer Splitter 3 times.After to be rinsed, 1mL buffer is added, the emigrated cells splitter from magnetic frame is quickly pushed with needle column, Go out the cell combined in splitter with one magnetic bead of CDl4 antibody, the as macrophage of CDl4+.

In addition, following 2 kinds of methods sorting, including 1. adherent method also can be used: PBMC being placed in and is cultivated containing RPMI-1640 In the culture dish of base, in 37 DEG C, contain 5%CO: cell incubator (Themo electro corporation CLASS 100, The U.S.) in be incubated for 2h.It after adherent mononuclear cells, inhales and abandons upper layer suspension cell, PBs buffer gently washs 3 times, is added a small amount of Mono- 1640 culture medium of RPMI, scrapes attached cell with cell scraper.1000r/min is centrifuged 5min, abandons supernatant.2. flow cytometry Method: CDl4 label: taking PBMC, is adjusted with buffer (bovine serum albumin(BSA) 2.5mL and the 2mol/LEDTA 0.5mL containing 10%) Whole cell density is 1 × 10⁸CDl4+-FITC antibody 100uL is added in/mL in every milliliter of cell suspension, and 4 DEG C are protected from light label 18min, then 1mL streaming buffer is added to terminate dyeing into centrifuge tube, PBs is washed 3 times, with the PBS for containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation in flow cytometer (BD FAcsAria II, the U.S.) on sort, according to the fluorescence intensity of CDl4 antibody, the relative particle of the relative size of cell and cell and interior The complexity of portion's structure collects the cell of CDl4+.

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

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

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

(3) CDl4 cell (macrophage) to be hybridized prepares: the mononuclear macrophage that the present invention sorts is with basal medium Total cell number is adjusted to 1 × 10⁸~2 × 10⁸For cell fusion.Blue dyeing phase-contrast microscopy, viable count are expected with platform It should be higher than that 80% is qualified.

(4) cell fusion: CDl4 cell (mononuclear macrophage) and myeloma cell are added with 10: 1-5: 1 ratio In centrifuge tube, it is mixed evenly, 1000r/m is centrifuged 5min, discards supernatant, and it gently beats tube bottom to cell grainless and precipitates, weight It is 2 times multiple.Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up PEG3000 is added drop-wise in fusion pipe in 60s along tube wall while gently rotating centrifugal pipe, later trains the basis of the 25mL of preheating It supports base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal pipe during addition is then allowed to stand In 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T culture medium is added.It is inoculated into after appropriate mixing In 96 well culture plates, 37 DEG C are placed in, is cultivated in 5% CO2 incubator.

(5) the hybridoma mononuclear macrophage strain with phagocytic function is screened: cell grows feelings in 96 well culture plates of observation Condition, division can be grown by only having hybridoma after 7-10 days, discarded HAT culture medium at this time, replaced complete medium.Cell gram When grand growth area reaches 1/10 cell hole, culture supernatant is gone, selection has the training of the good hybridoma cell strain of growth conditions Hole is supported, position, the size of cell strain growth are marked under microscope, cell clone is drawn in the position of mark using sterile pipette tips and arrives New has in the culture hole of complete medium, and then successively doubling dilution to hole is counted below, and 37 DEG C, 5%CO2 incubator is interior to be cultivated One week or so, microscopically observation cell growth status, when cell clone is covered with to 1/10 or more hole floor space, take cell or Culture supernatant detects hybridoma macrophage (M φ) strain function.The specific method is as follows:

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

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

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

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

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

6, the amplification of hybridoma macrophage strain

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

7, the preparation (preparation for the treatment of AIDS organelle) of hybridoma macrophage strain organelle

By the hybridoma macrophage strain of (above-mentioned) preparation of the present invention, after being cleaned with physiological saline, then with 1000r/min from Heart 5min (low speed is centrifuged in short-term) takes cell precipitation to be packed into cylinder made of acrylate etc high-biocompatibility material and describes In device, sterile saline is then filled it up with, makes the concentration 80%~90% of hybridoma macrophage strain, is sealed, is made immediately The treating AIDS organelle used.Hybridoma macrophage strain in organelle, which has, to be swallowed and adsorbs HIV and secrete huge The function of the phagocyte factor, the shape of organelle can be made into infundibulate, and bottom diameter is small, and top diameter is big, and volume is 200~300ml, disengaging Mouth is equipped with cell screen clothes, and entrance top diameter sieve mesh number is 800 mesh；Exit bottom diameter sieve mesh number is 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, The specification of 4.0 mesh, 4.5 mesh and 5.0 mesh, to stop 120 nanometers inhibition of HIV or bigger bacterium；Liquid outlet setting Mesh number is the cell strainer of 100 mesh (being equivalent to 4 microns), to the cell for stopping to filter out；Liquid entrance and mesh screen it Between be equipped with buffer area, be conducive to the stability of system circulation.When the blood plasma separated through extracorporeal circulation apparatus flows through organelle, trip From HIV by corresponding hybridoma macrophage strain swallow adsorb, purified blood plasma from organelle flow out, separated with separator Haemocyte converge after feed back it is internal.Treating AIDS organelle of the invention can entrust professional businessman to prepare.

8, the preparation of plasma separator

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

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

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

9, the application for the treatment of AIDS organelle

By the application schematic diagram of Fig. 1 in the present invention, plasma separator and organelle are connected, constitutes extracorporeal circulation apparatus, tool Body method is as follows:

(1) it installs: with sterile working connecting components, including plasma separator, organelle and each circulation line.

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

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

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

(5) start: venous line (5) being connected to the vein blood vessel of AIDS patient, then open blood pump, blood flow is 100~150ml/min, such as Fig. 1 are separated when arterial blood enters plasma separator (4) through arterial blood line pipe (1) Blood plasma reaches organelle (8) through circulation line (7) under the action of blood plasma pump 6, wait be full of blood plasma, about 10 minutes, begins releasing Blood plasma is flowed out through circulation line (10), synchronous that blood plasma is perfused to organelle (9), and the blood plasma in organelle (8) has nearly flowed When, perfusion blood plasma is started again at, organelle (9) begins releasing blood plasma at this time, and two organelles (8) in parallel, organelle (9) are handed over For progress.Such as Fig. 2, when blood to be separated enters inner cavity (2) of plasma separator (1), the effect through valve (8) can lead to The small molecule blood plasma components (5) for crossing micropore (3) enter the exocoel (6) of separator, then flow out through plasma outlet port (7), and cannot It is flowed out by the haemocyte (4) of micropore (3) through valve (8).Such as Fig. 3, when the blood plasma containing HIV (3) enters organelle (1), HIV (3) therein be fixed on the hybridoma macrophage strain (2) in organelle phagocytosis absorption, formed compound (4) and no longer It moves down, and the cell factor (5) that hybridoma macrophage strain generates is blended in the blood plasma after removing HIV and flows out cell After device, fed back through venous line shown in FIG. 1 (5).So until the plasma circulation amount (usually 9L) being previously set, treatment is It ends.If mating computer program control, entire therapeutic process is controlled by computer, and can detect working condition at any time, , automation more convenient using meeting and safety.

10, the additional component for the treatment of AIDS organelle

The treating AIDS organelle that Fig. 1 is provided is basic composition part of the invention, can also be reequiped on this basis With lower component, including sound pulse pressure and air monitering, temperature control system, liquid mixing system, off gas system, monitored conductivity system The parts such as system, ultrafiltration monitoring and leakage blood monitoring.

(1) sound pulse pressure monitors: in addition the main stopping state to dynamic monitoring organelle micropore of arterial blood pressure monitoring is used To monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When blood flow deficiency, angiosthenia will be reduced；When having blood coagulation, thrombus When formation, especially separator blockage of the micro orifice, angiosthenia will be increased；Vein pressure monitoring is used to monitor the pressure of pipeline blood reflux Power, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow deficiency and venous return syringe needle fall off, vein pressure will Decline, if bloody path return pipe distortion blocking or reflux syringe needle block, vein pressure will be increased.

(2) air monitering (Air Detector): the air bubble for monitoring blood pathway generally uses ultrasonic listening Principle, in order to avoid patient occur air embolism and be arranged.When having monitored air bubble, detection system can drive it is dynamic, Vein bloody path folder carrys out blocking blood flow, prevents dangerous generation.

In short, on the basis of basic component system of the present invention, it is expected to further research and develop automation therapeutic equipments, develops and be The hommization of operation, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily Judge the micro computers processing system such as alarm reason and ring off signal.

11, experimental verification

The effect of to understand effect of the invention, devising Simple cell device test method: take 2.5 × 300mm of sterilizing Westergren's blood sedimentation tube 5, draw respectively be centrifuged (1000r/min, 5min) precipitating the strain of hybridoma macrophage to 200mm carve Degree then draws the heat preservation after 100 DEG C dissolve and reaches about 10mm long scale in 56 DEG C of 0.9% spare agarose C1-4B, set After blood sedimentation stand is cooling, agarose becomes semisolid, and blood sedimentation tube inner cell can be prevented to flow out but not prevent water and the change of small molecule The substance for studying part etc passes through.5 blood for AIDS (AIDS) patient that Disease Control and Prevention Center of the Zhejiang Province Ling Qu equal samples library saves Slurry, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and injects blood sedimentation tube (Simple cell device) upper end blank pipe, blood to be flowed through in batches The hybridoma macrophage strain layer of immersed tube lower layer simultaneously after outflow, collects efflux, blood plasma after referred to as AIDS filter out of blood sedimentation tube.It takes Blood plasma and blood plasma after filter before AIDS is filtered, with human macrophage migration inhibitory factor (MIF) ELISA detection kit (hundred stamen of Shanghai Biotechnology Co., Ltd) pairing detection, by specification operation, detection range is 0~800pg/ml, susceptibility 1.0pg/ Ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive stronger, and negative reaction is colourless or pole Shallowly, it according to the depth of be in color, is indicated with "+", "-" number.OD value can also be surveyed: on ELISA detector, in 450nm (if with ABTS develops the color, then 410nm) at, each hole OD value is surveyed after returning to zero with blank control wells, if more than defined negative control OD value It is 2.1 times, as positive.As a result such as table 1, MIF testing result is negative (or because content is sensitive lower than detecting in blood plasma before filtering Degree, blood plasma long-term preservation cause degradation etc.), and testing result is the positive in blood plasma after filtering, and illustrates macrophage hybridoma Strain produces MIF cell factor in this process.MIF is to integrate cell factor, growth factor, the multiple-effect of hormone and enzyme characteristic Can protein molecular, the effect of central is played as inherent immunity and the regulatory factor of inflammatory reaction, in various infection and anxious slow Property diseases associated with inflammation in play panimmunity function.MIF pairing detection is same before and after making AIDS blood plasma filtration Simple cell device When, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 antigen detection kit (enzyme-linked immunization, Shanghai Inspire Biotechnology Co., Ltd) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, As control, minimum detection limit is lower than 5pg/ml for the p24 antigen of 20pg/ml, 40pg/ml, 80pg/ml, and measurement range 0~ 450nm measures absorbance (OD) in 400pg/ml, the range of linearity 0.5pg/ml~80pg/ml, 15min, blank control calibration object Absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbance not higher than 0.050,0pg absorbance value, works as extinction Be considered as when spending > 0.12 it is positive, after testing result (table 2) illustrates AIDS blood plasma filtration Simple cell device, part HIV by Hybridoma macrophage strain phagocytosis absorption, the blood plasma HIV after filtration are significantly reduced, and after the 1st filtration, HIV clearance rate is 20.55%, after the 2nd filtration, HIV clearance rate is 42.83%, p < 0.01, has significant effect, illustrates with filtration The increase of number, HIV can be removed constantly, to reach treatment AIDS purpose.

MIF pairing testing result is (quantitative: pg/ml) before and after 1 AIDS blood plasma of table filters Simple cell device

2 AIDS blood plasma of table filters p24 testing result (p24:pg/ml) before and after Simple cell device

In short, from Tables 1 and 2 the result shows that, the blood plasma containing HIV is filtered through treating AIDS organelle of the invention Afterwards, HIV therein can be adsorbed by the hybridoma macrophage strain in organelle and be removed, significant effect；At the same time, hybridoma is huge Phagocyte strain is produced with anti-infective and immunization cell factor.The experiment results show that treating AIDS of the invention Organelle has significant effect.

Claims (13)

1. a kind for the treatment of AIDS organelle for field of biomedicine, which is characterized in that including high-biocompatibility material And hybridoma cell strain, the hybridoma cell strain is wrapped up with high-biocompatibility material, is constructed to prevent cell and its fragment And HIV filters out and can swallow for hybridoma cell strain HIV and generate cell factor and provides the organelle in place；With primary cell Cd4 cell therein is sorted, after cultivation, CD14 cell is further sorted, is followed by prepared into the CD14 cell sorted both Retain archaeocyte characteristic and the hybridoma cell strain of energy infinite multiplication, the hybridoma cell strain can swallow HIV and generate thin Intracellular cytokine.

2. treating AIDS organelle according to claim 1, which is characterized in that the primary cell includes single core blood Liquid cell, single core histocyte, amniocyte and/or villus cell.

3. treating AIDS organelle according to claim 1, which is characterized in that the method for sorting CD14 cell includes exempting from Epidemic disease paramagnetic particle method, adherent method, flow cytometry.

4. treating AIDS organelle according to claim 2, which is characterized in that the method for sorting CD14 cell includes exempting from Epidemic disease paramagnetic particle method, adherent method, flow cytometry.

5. treating AIDS organelle according to claim 1, which is characterized in that the hybridoma cell strain is by myeloma Cell SP2/0 or human myeloma cell strain are prepared.

6. treating AIDS organelle according to claim 2, which is characterized in that the hybridoma cell strain is by myeloma Cell SP2/0 or human myeloma cell strain are prepared.

7. treating AIDS organelle according to claim 3, which is characterized in that the hybridoma cell strain is by myeloma Cell SP2/0 or human myeloma cell strain are prepared.

8. treating AIDS organelle according to claim 4, which is characterized in that the hybridoma cell strain is by myeloma Cell SP2/0 or human myeloma cell strain are prepared.

9. -8 any treating AIDS organelle according to claim 1, which is characterized in that the volume of organelle is 200 ~300ml, is equipped with top diameter cell screen clothes at liquid-inlet, and top diameter cell screen clothes mesh number is 800 mesh, liquid-inlet with push up diameter cell The buffer area that promotion system stablizes circulation is equipped between sieve, exit is equipped with bottom diameter cell screen clothes and cell strainer, and bottom diameter is thin Born of the same parents' sieve mesh number is 2.0~5.0 mesh, and cell strainer mesh number is 100 mesh, is also equipped between bottom diameter cell screen clothes and liquid outlet slow Area is rushed, the stability of system circulation is conducive to.

10. treating AIDS organelle according to claim 9, which is characterized in that organelle exit bottom diameter sieve mesh Numeral system at 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh 7 kinds of different sizes.

11. -8,10 any treating AIDS organelle according to claim 1, which is characterized in that the organelle and blood It starches separator to be connected, the plasma separator is hollow fibre type, and diameter is 270~370 μm, film thickness is 50 μm, aperture is 0.2~0.6 μm, length be 13.5~26 μm, can stop every other blood constituent, only allowance blood plasma filter.

12. treating AIDS organelle according to claim 9, which is characterized in that the organelle and plasma separator It is connected, the plasma separator is hollow fibre type, and diameter is 270~370 μm, film thickness is 50 μm, aperture is 0.2~0.6 μ M, length is 13.5~26 μm, can stop every other blood constituent, only permits blood plasma filtration.

13. any treating AIDS organelle of claim 1-12 is preparing the application in extracorporeal blood circulating device, It is characterized in that, the extracorporeal blood circulating device includes arterial blood line pipe (1), one end is through heparin pump (2) and blood pump (3) It is connected with plasma separator (4), plasma separator (4) first cell in parallel with two through blood plasma pump (6) and circulation line (7) Device (8), the second organelle (9) are connected, and are then successively connected with circulation line (10), venous line (5).