CN106110425B - AIDS plasma purification therapeutic equipment - Google Patents
AIDS plasma purification therapeutic equipment Download PDFInfo
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- CN106110425B CN106110425B CN201610540910.8A CN201610540910A CN106110425B CN 106110425 B CN106110425 B CN 106110425B CN 201610540910 A CN201610540910 A CN 201610540910A CN 106110425 B CN106110425 B CN 106110425B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3692—Washing or rinsing blood or blood constituents
Abstract
A kind of AIDS plasma purification therapeutic equipment for medical domain, it is characterized in that preparation energy separated plasma, the blood and plasma separator of mononuclear blood cell and the multinucleate giant cell formed because living away from home HIV, preparation not only retains phagocytosis characteristic but also the hybridoma macrophage strain of energy indeterminate growth, cells frozen storing liquid is formulated in after amplification, make cell concentration up to 80%, then clarifier made of high molecular material is perfused, macrophage strain therein plays a part of to swallow HIV, clarifier and blood, plasma separator constitutes crucial extracorporeal circulation apparatus, when blood stream is through blood separator, multinucleate giant cell containing HIV is filtered out, and then when flowing through clarifier by the blood plasma that plasma separator separates, HIV therein is cleaned agent removing, the purified blood plasma mononuclear blood cell separated with plasma separator is fed back after converging , to reach the purification treatment purpose for removing the inside and outside HIV of haemocyte.
Description
Technical field
The present invention relates to the preparations and application of AIDS plasma purification therapeutic equipment in medical domain, are mainly used for AIDS patient
The removing of the inside and outside AIDS virus of haemocyte, to achieve the purpose that treat AIDS.
Background technique
It after HIV enters human body, is swallowed first by macrophage, but HIV changes certain positions in macrophage quickly
Acidic environment creates the condition for being suitble to it to survive, is not killed not only and breeds in it instead.Because CD4 be HIV by
Body, so the HIV bred in macrophage is through its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge
Effect, utilizes itself hydrophobic effect mediate retroviral cyst membrane and cell membrane fusion) entering CD4+ cell, (cell, monokaryon macrophage are thin
Born of the same parents, Dendritic Cells etc.), it is proliferated rapidly in the cell, generates 10 daily9~1010Virion, and it is normal constantly to enter other
And regenerated CD4+ replicate into the cell, manufacture more virus infected cells, make peripheral blood CD4+T cell sustaining breakdown, subtract
It is few.The T cell of infected Apoptosis or even infected by HIV can be directly activated to express in conjunction with gp120 and the CD4 receptor of HIV
Envelope antigen can also start normal T-cell, cause a large amount of broken of CD4+ cell indirectly by cell surface CD4 molecule cross-link
Bad, as a result cause the severe immune deficiency centered on CD4+T cell defect, patient mainly shows: periphery lymphocyte is reduced,
T4/T8 proportional arrangement disappears to the reaction of phytohemagglutin phytolectin and certain antigens, delayed allergy decline, NK cell, macrophage
Cell activity weakens, and the synthesis of the cell factors such as IL2, interferon is reduced.CD4+T cell is most important immunocyte, infection
Person once loses a large amount of CD4+T cells, and entire immune system will all lose the infection of various diseases by deathblow
Go resistance.HIV can also show as hiding without showing clinical symptoms, genome for a long time after entering host's CD4+ cell
RNA reverse transcription enters in host cell nuclear at double-stranded DNA with viral integrase enzyme, under the action of integrase, double-stranded DNA integration
Into host cell gene group, the viral DNA being integrated is known as provirus, and the several months that can hide does not replicate even for many years, causes
The incubation period of AIDS several months to many years.In the incubation period of AIDS, HIV is mainly in the macrophage and Dendritic Cells of lymph node
Breeding, these cells are intracorporal HIV depots, and releasably the CD4+T cell into peripheral blood or transfection peripheral blood, there is silk point
Splitting original, antigen, TNF, IL-2 and lymph plain (LT) can excite HIV provirus gene multiple in the CD4+T Intracellular transcription of infection
System.After being largely proliferated, inhibition of HIV particle constantly discharges from the infection cell being destroyed and is free on blood, then enters back into
Other cells continue course of infection.
Body production envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody can be stimulated after HIV infection.?
Low-level antiviral neutralizing antibody is measured in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum,
HIV carriers' highest illustrates that the antibody has protective effect in vivo.But antibody cannot be with the virus that retains in mononuclear macrophage
Contact, and antigenic variation easily occurs for HIV envelope protein, original antibody is ineffective, keeps neutralizing antibody due from playing
Effect.In the latent infection stage, HIV provirus is integrated into host cell gene group, therefore HIV will not be known by immune system
Not, so relying solely on itself immune function can not be removed.Another critically important reason should be killed according to antibody
It goes out, remove the mechanism speculate of antigen, after immune antibody and antigen binding, if to generate immunological effect or pass through activation
Complement mediates ADCC effect to dissolve cellular antigen, but HIV is not cellular antigen；Attracted by chemotaxis and is swallowed
Antigen is removed in cell phagocytosis, but HIV is protected in phagocyte instead, is proliferated；Antibody and antigen binding, which rise to neutralize, to be made
With being allowed to lose appeal, but HIV antigenic structure is changeable, is often difficult to antibody.
In terms of the treating AIDS method for having been used for clinic at present, effect is less ideal: (1) hiv reverse transcriptase inhibits
Agent: being only capable of preventing the permissive cell of not yet infected by HIV from infecting, and does not have a therapeutic effect to the cell infected, and toxic side effect compared with
It is more, including gland plastochondria toxicity, bone marrow suppression, globular anemia, granulocyte and decrease of platelet, pancreatitis, intersect in addition resistance to
The generation of pharmacological property, this kind of medicine are not used alone, mainly do drug combination, and are also easy to produce drug resistance mutant, cause under clinical efficacy
Drop or failure.(2) toxic side effects such as drug induced hepatic injury, disorders of lipid metabolism and drug resistance hiv protease inhibitor: are also easy to produce
Property.(3) it hiv integrase inhibitor: by inhibiting inhibition of HIV DNA to play a role with host cell DNA integration, is reversed with HIV
Transcriptase inhibitors and protease inhibitors are combined while use has synergistic effect to antiviral.(4) inhibition of HIV is inhibited to enter inhibition
Agent: including block gp120 with CD4 ining conjunction with, blocking HIV in conjunction with accessory receptor, act on gp41 film subunit and act on T drench
Bar cell surface CC-chemokine receptor 5 (CCR5) has side effect to liver and heart to block HIV to enter host cell.(5)
Cytokine therapy: it is mainly used for regulatory T-cell dynamic equilibrium.(6) HIV vaccine is treated: such as intrinsic due to the particularity of HIV
Immune to be not enough to resist HIV and its targeting destruction immune system, virus mutation is rapid, causes not yet to develop real safety so far
Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, including antisense technology, RNA bait, RNA dry
It disturbs, intrabody, dominant negative mutant, suicide gene etc., but enters the gene therapy in II clinical trial phase stage almost
No.(8) monoclonal antibody passive immunization therapy: the neurological susceptibility of HIV is reduced by lowering CD4+T cell surface CCR5, is prolonged
The progress of slow AIDS and the diffusion for reducing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10, which are applied to HIV infection person, to be had
Good tolerance and safety can delay but cannot prevent rebound (9) adoptive immunity cell therapy of virus: external a large amount of
It will lead to viral massive amplification when the culture self CD4+T cell of HIV, increase the CD4+T cell quantity of virus infection, and feed back
CD4+T cell may will increase the place of internal virus replication, lead to virus load bounce-back, and on the whole, adoptive immunity is thin
Born of the same parents treat without apparent toxic side effect, also do not obtain satisfied therapeutic effect.
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.So can be with conventional hybridization
Oncocyte technology of preparing, prepares macrophage hybridoma, and the clarifier for the treatment of AIDS is used to prepare after massive amplification,
The HIV in blood plasma is removed with the phagocytic function of macrophage.
In short, various drugs and biological products can not effectively kill intracorporal AIDS virus, and price, side effect is big,
So far the effective ways for the treatment of AIDS be there is no, it has also become attack the global problem being unable to long.
Summary of the invention
In order to solve to attack the global problem in the treating AIDS field being unable to long, present inventors have proposed the present invention.
The invention aims to provide AIDS plasma purification therapeutic equipment；Another object is to provide for AIDS plasma purification and controls
Treat preparation and the application method of instrument.
The object of the present invention is achieved like this: it is thin that preparation can filter out the large volume containing HIV that many cells are combined into
The blood separator of born of the same parents and the plasma separator that can separate mononuclear blood cell and blood plasma had both been retained former huge with hybridoma technology preparation
The hybridoma macrophage strain of phagocyte characteristic and energy indeterminate growth expands parallel, and macrophage strain is taken to be formulated in cell cryopreservation
Liquid makes cell concentration up to 80%, and being then perfused made of high-biocompatibility material can prevent cell and its fragment from filtering out simultaneously energy
HIV being swallowed for cell strain, the plasma purification device in place being provided, wherein macrophage strain is fixed in plasma purification device, plays phagocytosis
HIV is used, made AIDS plasma purification device merge additional computer regulatory process with blood, plasma separator group in turn and
AIDS plasma purification therapeutic equipment is prepared, the blood separator that the blood in extracorporal circulatory system is treated in instrument filters out the multicore of large volume
Giant cell, and then blood plasma and mononuclear blood cell are divided by plasma separator, the purified device of blood plasma remove after HIV with mononuclear blood cell
Converge feedback.
The present invention is made 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 out HIV infection cell in blood
The multinucleate giant cell of formation or many cells condensate, plasma separator can separate the mononuclear blood cell and blood plasma of medium volume, blood
The cleanser starched in clarifier is macrophage strain, is formulated in after cells frozen storing liquid and saves backup convenient for prolonged cold, because of macrophage
The CD4 molecule of cell expression is the receptor of AIDS virus, has the characteristic of natural phagocytosis HIV, and the present invention simulates macrophage and exists
The mode of non-specific phagocytosis in vivo, when the blood plasma containing HIV flows through clarifier, HIV and macrophage be in contact and by
Phagocytosis, is detained in clarifier therewith, so containing a large amount of HIV's in the extracorporal circulatory system of AIDS plasma purification treatment
Large volume cell is filtered out by blood separator first, and the HIV to dissociate in blood plasma is removed by plasma purification device, purified blood plasma
It is fed back after converging with the mononuclear blood cell of medium volume, is incorporated in cell surface and/or intracellular HIV and trip to remove
From in the HIV of blood plasma.The present invention can not effectively kill the conventional internal of HIV to filter out the method substitution of HIV in vitro for a long time
Anti-reverse transcription drug treatment.
Specific embodiment
Fig. 1 is the application schematic diagram of the AIDS plasma purification therapeutic equipment proposed according to the present invention.
Fig. 2 is the schematic diagram of internal structure of the blood separator proposed according to the present invention.
Fig. 3 is the schematic diagram of internal structure of the plasma separator proposed according to the present invention.
Fig. 4 is the schematic diagram of internal structure of the clarifier proposed according to the present invention.
In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, the other end through heparin and blood pump (2) with contain
There is the blood separator (3) of waste liquid outlet (5) to be connected, blood separator (3) exports (4), blood pump (6), circulation pipe through blood
Road (7) is connected with plasma separator (8), and the plasma outlet port of plasma separator (8) is through blood plasma pump (9) and blood vessel (10) and two
Clarifier (11) in parallel is connected with clarifier (12), the export pipeline (13) of two clarifiers and the blood of plasma separator (8)
Cell outlet pipeline (14) converge after through venous line (15) converge body circulation.
Fig. 2 indicates the internal structure of the blood separator (3) in Fig. 1.In Fig. 2, the tube wall of the inner cavity (2) of separator (1)
On have many micropores (3), multinucleate giant cell (4) cannot filter micropore (3) and be delayed at inner cavity (2), so as to be removed, energy
By in micropore (3), the mononuclear blood cell (5) of small size enter exocoel (6), (7) outflow is then exported, and then through Fig. 1
Shown in plasma separator (8) separation blood cells and blood plasma.
Fig. 3 indicates the internal structure of the plasma separator (8) in Fig. 1.In Fig. 3,1 is plasma separator, and 2 be blood plasma separation
Device inner cavity, 3 be the micropore on the tube wall of plasma separator inner cavity, 4 be cannot be by the mononuclear blood cell of micropore (3), 5 be that can pass through
The blood plasma chemical analysis of micropore (3), 6 be plasma separator exocoel, and 7 be blood plasma outflux, and 8 be to have the blood of switchable valve thin
Born of the same parents outlet.
In Fig. 4,1 is clarifier, and 2 be the macrophage strain in clarifier；3 be the HIV for entering clarifier together with blood plasma,
4 are cleaned the phagosome that no longer moves down after the phagocytosis of the phagocyte in device for HIV, 5 macrophages generated for macrophage because
Son.
Below with reference to Fig. 1, Fig. 2, Fig. 3 and Fig. 4, the embodiment of AIDS plasma purification therapeutic equipment proposed by the present invention 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 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
RPMI-1640 culture medium 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 mononuclear macrophage strain with phagocytic function is screened: cell growth status in 96 well culture plates of observation, 7-10
Division can be grown by only having hybridoma after it, discarded HAT culture medium at this time, replaced complete medium.Cell clone growth
When area reaches 1/10 cell hole, culture supernatant is gone, selection has the culture hole of the good hybridoma cell strain of growth conditions, shows
Position, the size that cell strain growth is marked under micro mirror, draw cell clone in the position of mark using sterile pipette tips has to new
In the culture hole of complete medium, then successively doubling dilution to hole is counted below, and 37 DEG C, the interior culture of 5%CO2 incubator one week left
The right side, microscopically observation cell growth status take in cell or culture when cell clone is covered with to 1/10 or more hole floor space
Clear detection hybridoma macrophage (M φ) strain function.
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, hybridoma macrophage strain treatment cell preparation
That is the amplification cultivation 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 macrophage hybridoma cell strain, see whether steady
It is fixed.Continuation carries out extensive industrialization preparation in several bottles, saves backup.
Two, the preparation of AIDS blood purification
1, the filling of cleanser
By hybridoma macrophage strain prepared by the present invention, after being cleaned with sterile saline, then it is centrifuged with 1000r/min
5min (low speed is centrifuged in short-term) takes cell precipitation assembly acrylate etc high-biocompatibility material (with plasma separator material
Expect identical) made of cylindrical cleaner, until 4/5, then plus cells frozen storing liquid (contains 30% fetal calf serum, 12% dimethyl sulfoxide
1640 culture medium), make cell concentration up to 80%, gently shake up, seal, through 4 DEG C, 0.5h；- 20 DEG C, 2h；- 70 DEG C, overnight,
It is spare to enter -196 DEG C of liquid nitrogen cryopreservations.It thaws in use, needing rapidly to put into cryopreservation tube in 37 DEG C of water-baths having been warmed up, not
It is disconnected to shake, melt the liquid in pipe rapidly, is used after then being cleaned with sterile saline.
2, the specification of clarifier
Clarifier can be the cylinder or rectangular, infundibulate that bottom diameter is small, top diameter is big, and volume is 200~300ml, inlet and outlet
Be equipped with cell screen clothes, entrance top diameter sieve mesh number is 800 mesh: exit bottom diameter sieve mesh number be 2.0~5.0 mesh (2.5~
5.0 mesh are equivalent to 0.1~0.2 micron or 100~200 nanometers), it 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, to stop 120 nanometers inhibition of HIV or bigger bacterium；Liquid outlet
The cell strainer that mesh number is 100 mesh (being equivalent to 4 microns) is set, to the cell for stopping to filter out；Liquid entrance and net
It is equipped with buffer area between sieve, is conducive to the stability of system circulation.
3, the material of clarifier
Blood purification selects the high molecular material of acrylate etc, it is desirable that good biocompatibility, hardly activation are mended
Body, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.Can by covalently,
The methods of grafting, polymerization improve the structure of material, the inhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxidative stress
Influence, to improve biocompatibility, reduce complication generation.Add hydrophilic gel in absorber inner surface, by 2 methyl-props
Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate film, by controlling wet-spinning procedure, is produced
CA/PMB30, CA/PMB80 and CA/PMB30-80, blood and cell compatibility with higher.There is anticoagulation by certain
Substance be solidificated on the material of carrier or absorber inner surface, can inhibit blood clotting, improve biocompatibility, can also reduce
Heparin dosage, and be possible to realize no-rod tractor.Heparin covalent is integrated to polyether sulfone surface, has both maintained the mechanics of polyether sulfone
Performance, and the anticoagulation function of absorber inner surface can be improved.The covalent immobilisation linoleic acid film on cellulose acetate film, or will be covalent
The linoleic acid for being integrated to polyacrylic acid is grafted onto polysulfones film surface, can there is better histocompatbility and anticoagulant effect.
With the continuous development of high molecular material and nanotechnology, close material will go out in the near future with Human vascular endothelial
It is existing.
Three, the preparation of separator
(1) preparation of blood separator
1, preparation principle: 1. haemocyte, bacterium, virus molecular size: visible component (cell) is big in blood of human body
It is small are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, and neutrophil leucocyte is about
12 μm, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, and 6-8 μm of small lymphocyte, with red blood cell
Approximation, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, and 1~4 micron to 7~8 microns of diameter is differed, the blood of people
Platelet average diameter is 2-4 microns, 0.5~1.5 micron thick.The size of bacterium are as follows: the diameter of coccus about 0.75-1.25 μm it
Between, for bacillus length about at 2-5 μm, spirillum is about 100-200 μm.The size of virus is with nanometer (nm) for unit [1cm=
10mm, 1mm=1000 μm, 1 μm=1000nm], difference in size is very big between different virus, the smallest gemnivirus such as plant
(Geminiviruses) diameter only 18-20nm, maximum animal poxvirus (Poxviruses) size up to 300-450nm ×
170-260nm, it is longest if filamentous virus section (Filoviridae) virion size is 80nm × 790-14000nm, majority
For the diameter of single virus particle in 100nm or so, AIDS virus is 100-120nm (0.1-0.12 μm).2. AIDS patient's blood
Related compounds present in liquid: multinucleate giant cell is (made of gp120 and the CD4+ cell combination of HIV infection cell surface substantially
Long-pending HIV infection cell), gp120 cell (there is gp120 on surface but with HIV infection cell existing for individual cells), gene integration
(HIV infection initial stage or incubation period are integrated with HIV double-stranded DNA to cell, but cell surface does not have the HIV infection of gp120 thin
Born of the same parents), normal white cell (being uninfected by granulocyte, monocyte existing for the individual cells of HIV, lymphocyte), red blood cell, blood it is small
Plate, chemical analysis (protein, carbohydrate, lipid, electrolyte etc.), free HIV, bacterium and other microorganisms.3. multicore is big and small
Born of the same parents are natural large volume cell；Gp120 cell and gene integration cell can make cell by the immune response of antigen and antibody
Agglutination is large volume many cells condensate；Free HIV can be changed into large volume composition by carrier granular/immune response.④
According to above-mentioned 3 points, can prepare can be by individual cells but cannot be by large volume cell or the blood separator of particle.5. selecting
The material of the selective adsorption function of apparatus screens out the HIV infection cell in blood through extracorporeal circulation of blood branch of the invention.
2, it the material of blood separator and requirement: with clarifier of the invention, selects poly-vinegar non-woven fabrics, acetate fiber, take off
Rouge cotton etc., it is desirable that good biocompatibility, hardly activating complement do not cause inflammatory reaction and leucocyte, blood platelet, blood oxygen point
The change of pressure, C3a, C5a.
3, the model of blood separator: the shape of blood separator is prepared into column construction (with acetate fiber or absorbent cotton
For material make filter core), flat structure (using poly-vinegar non-woven fabrics be material production filter core)；Aperture is prepared into 150~250 μm, 50
~150 μm, 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm, 1~2 μm of model.
4, the separator of different model, principle the application of principle of blood separator: are selected according to the state of an illness of AIDS patient
It is upper first large aperture model to be selected to do pre- sieving, then select the model of smaller aperture due.Severe AIDS patient often occurs serious
Opportunistic infections contain different size of composition in blood.Such as containing the fungi of especially big volume, spirillum, tumour cell and its
His foreign matter, then selecting aperture is 150~250 μm or 50~150 μm of separator；The monokaryon macrophage of for example sieving HIV infection is thin
Born of the same parents, multinucleate giant cell, many cells condensate and the particulate matter for being adsorbed with HIV, and in order to replace vulnerable to HIV infection
CD4+ cell then selects 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm etc of model.These types of model is approximate or is less than
The volume of single red blood cell, neutrophil leucocyte, small lymphocyte in blood, but red blood cell, neutrophil leucocyte and macrophage tool
There is the characteristic of amoeboid movement, micropore more smaller than own vol can be passed through.
(2) preparation of plasma separator
(1) it preparation principle: is prepared according to the molecular size of haemocyte and blood plasma components.Such as visible component in blood of human body
The size of (haemocyte) are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds,
About 12 μm of neutrophil leucocyte, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, small lymphocyte 6-
8 μm, approximate with red blood cell, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, 1~4 micron to 7~8 microns of diameter
It differs, the platelet mean diameter of people is 2-4 microns, 0.5~1.5 micron thick.
(2) poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. material: can be selected, it is desirable that good biocompatibility hardly swashs
Living complement, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.It can be by altogether
The methods of valence, grafting, polymerization improve the structure of material, the microinhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxygen
Change stress influence, the generation to improve sieving adequacy and biocompatibility, reduce complication.
(3) type and spec: for the shape of separator, filter core preparation can be made with materials such as acetate fiber or absorbent cotton
The shapes such as flat structure are prepared into as filter core at column construction, with materials such as poly-vinegar non-woven fabrics；By haemocyte and blood to be separated
The molecular size of slurry composition determines aperture.It is plasma separator according to the present invention property stabilization, good biocompatibility, penetrating
Property high high molecular polymer hollow fibre type filter is made, hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm,
Aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm.The hole only permit blood plasma filtration, but can stop all cells at
Point.
Four, the component of AIDS plasma purification therapeutic equipment
1, key member: (1) blood separator: for screening out by volume size with multinucleate giant cell or many cells condensate
HIV infection cell existing for state, i.e., for removing endoglobar HIV；(2) plasma separator: thin for separating single blood
Born of the same parents and blood plasma；(3) plasma purification device: for the HIV in adsorbed plasma.
2, additional member: including blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system,
The parts such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring form.(1) blood pump (Blood Pump): for pushing blood
Circulation is to maintain going on smoothly for blood purification treatment, and usual blood pump part often has rotary test speed function, to monitor patient
Blood circumstance, therefore blood pump runner and the setting of groove spacing are accurate, and need often adjustment, according to the feelings of bloody path pump line
Spacing is generally set as 3.2~3.3mm by condition, can not be too loose, and it is inaccurate otherwise to will cause blood flow detection；Also can not be too tight, otherwise
It will cause pipe breakage.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump clinically applied, and uses
It to continue the injecting heparin in sieving pipeline (patient blood), contacts, is easy with air since the blood of patient recycles in vitro
Blood coagulation phenomenon occurs, anticoagulative can be occurred using heparin pump.(3) sound pulse pressure monitor: arterial blood pressure monitoring mainly to
The stopping state of dynamic monitoring blood separator micropore, in addition to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When
When blood flow deficiency, angiosthenia will be reduced；When having blood coagulation, thrombosis, especially separator blockage of the micro orifice, angiosthenia will
It increases；Vein pressure monitoring is used to monitor the pressure of pipeline blood reflux, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow
When insufficient and venous return syringe needle falls off, vein pressure will decline, if bloody path return pipe distortion blocking or reflux syringe needle
When blocking, vein pressure will be increased.(4) air monitering (Air Detector): the air gas for monitoring blood pathway
Bubble, the general principle for using ultrasonic listening, in order to avoid air embolism occurs for patient and is arranged.When having monitored air bubble
When, detection system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent dangerous generation.
In short, Import computer regulates and controls and is made the people of operation on the basis of key member of the present invention and additional member
Property, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily judgement is warned
Report the blood purifying therapeutical instrument of the micro computers such as reason and ring off signal processing.
Five, the connecting path and application method of AIDS plasma purification therapeutic equipment
1, it installs: such as Fig. 1, with sterile working connecting components, including blood separator, plasma separator, plasma purification
Device and each circulation line.
2, it is vented: with sterile saline filling liquid separator, clarifier and each circulation line, excluding separator, clarifier
And its gas, bubble in circulation line, it goes through, confirmation after gas, bubble without using.
3, lead to liquid: arterial blood line pipe (1) being connected to the arteries of AIDS patient, in operation the row of going through again
Whether gas is complete, and whether liquid stream is unobstructed, and flow liquid in pipe is avoided to pollute.
4, anticoagulant: to inject anti-coagulants (heparin) into liquid stream from heparin pump (2), be for the first time 2500 ∪ or 20~30 ∪/kg.
5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (15) are connected into venous blood
Pipe, then open blood pump (2), blood flow be 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 multinucleate giant cell formed by HIV infection is delayed at
In blood separator (3), mononuclear blood cell and blood plasma are successively flowed into through blood outlet (4), blood pump (6) and circulation line (7)
Plasma separator (8), isolated blood plasma successively flow into clarifier (11) open at this time through blood plasma pump (9) and blood vessel (10),
Wait be full of blood plasma, about 10 minutes, blood plasma is begun releasing, is flowed out through export pipeline (13), it is synchronous that blood plasma is perfused to clarifier (12),
When blood plasma in clarifier (11) has nearly flowed, perfusion blood plasma is started again at, clarifier (12) begins releasing blood plasma at this time, and two
The clarifier (11) of a parallel connection and (12) are alternately.Such as indicate Fig. 2 of the internal structure of the blood separator (3) in Fig. 1, blood
Have many micropores (3) on the tube wall of the inner cavity (2) of liquid/gas separator (1), multinucleate giant cell (4) cannot filter micropore (3) and be hindered
Inner cavity (2) are stayed in, so as to be removed, can be passed through in micropore (3), the mononuclear blood cell (5) of small size and blood plasma are outside
Chamber (6) is then exported (7) outflow, and then separates blood cells and blood plasma through plasma separator shown in FIG. 1 (8).As indicated
Fig. 3 of the internal structure of plasma separator (8) in Fig. 1 has many micropores on the tube wall of the inner cavity (2) of plasma separator (1)
(3), there cannot be the haemocyte of switchable valve outlet (8) outflow by the mononuclear blood cell (4) of micropore (3), into Fig. 1
Shown in haemocyte export pipeline (14)；Plasma separator exocoel can be entered by the blood plasma and its chemical component (5) of micropore (3)
(6), then enter clarifier through blood plasma outflux (7), blood plasma pump shown in FIG. 1 (9) and blood vessel (10).Such as indicate in Fig. 1
Fig. 4 of clarifier (11) and (12) internal structure, when the blood plasma containing HIV (3) enters clarifier (1), HIV (3) therein
The macrophage phagosome (4) no longer moved down is formed after macrophage (2) phagocytosis being cleaned in device, it is net after being removed HIV
Change the individual cells that blood plasma is separated through export pipeline shown in FIG. 1 (13) with plasma separator (8) to converge in export pipeline (14)
Converge by venous line (15).HIV is so removed, blood is purified, until the plasma circulation amount (usually 9L) being previously set,
Treatment just ends, and entire therapeutic process is controlled by computer, and can detect working condition at any time, easy to use, automation
And safety.
Six, the verifying of AIDS plasma purification therapeutic equipment effect
1, blood separator filters out the verifying of HIV infection cell effect
The present inventor's basic skills according to the invention has done following simple confirmatory experiment: having taken Disease Control and Prevention Center and infection
The anticoagulated whole blood several pieces for AIDS (AIDS) patient made a definite diagnosis that sick laboratory biological sample database saves, part phase of fetching respectively
Anticoagulated whole blood with abo blood group is mixed into 5, keeps blood volume sufficiently large, then entrusts hospital center of Zhejiang Province blood station proportionately
The blood component separation method of part blood transfusion, isolates leucocyte, red blood cell, blood plasma through blood component separation system, takes leucocyte
Composition routinely centrifugation inhales abandoning supernatant with suitable physiological saline suspension leukocyte cell pellet and proper ratio is then added
Gp120 antibody (Shanghai Guang Rui Biotechnology Co., Ltd), mix 37 DEG C of postposition react 5 minutes, then with aperture be 20~
The blood component separation system of 30um isolates the leucocyte (referred to as big leucocyte) of large volume, again to the leucocyte filtrate of filtration
The leucocyte (leucocyte in referred to as) of medium volume is further isolated with the blood component separation system that aperture is 15~25um,
Leucocyte in filtrate is the leucocyte (referred to as small white blood cells) of small size, collects large, medium and small leucocyte separation suspension respectively,
Conventional centrifugal precipitating, inhales and abandons supernatant, with the large, medium and small leukocyte cell pellet of quantitative liquid shifter difference draws equal amounts, conventional method
(mechanical or cell pyrolysis liquid) lytic cell (such as with lysate of the same race, needing dosage equal), takes supernatant, then after centrifugation
According to HIV-1p24 antigen detection kit (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known dense
Spend the p24 antigen conduct of 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml
Control, minimum detection limit are lower than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity 0.5pg/ml~80pg/ml, 15min
Interior 450nm measures absorbance (OD), and blank control calibration object absorbance value is not higher than not higher than 0.050,0pg absorbance value
0.100,1000pg/ml absorbance is not less than 1.000, is considered as the positive as absorbance > 0.12, and testing result (table 1) is said
Bright, the HIV-p24 content in the leucocyte of AIDS patient different volumes size is different, the HIV-p24 in large, medium and small leucocyte
Average content be respectively 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, wherein HIV-p24 is average in large and small leucocyte
Content differs 148.6pg/ml, reduces 54.4%；The total content of HIV-P24 is respectively in large, medium and small leucocyte
1375.0pg/ml, 979.9pg/ml, 632.1pg/ml, wherein HIV-p24 total content differs 742.9pg/ in large and small leucocyte
Ml reduces 54.3%, through statistical test, t=2.43, p < 0.05.Illustrate the intracorporal large volume leucocyte of AIDS patient or
HIV containing high level in the large volume leucocyte formed after the effect of gp120 antibody, can be by implementing skill of the invention
Art scheme is removed by separation.
HIV-p24 testing result (p24:pg/ml) in the 1 large, medium and small leucocyte of AIDS patient peripheral blood of table
2, clarifier removes the verifying of HIV effect
The effect of removing HIV for check cross tumor macrophage strain, the present invention devises easy test method: taking and goes out
2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5 draws the macrophage hybridization for being centrifuged (1000r/min, 5min) precipitating respectively
Tumor cell strain is then drawn and is kept the temperature after 100 DEG C dissolve in 39~41 DEG C of 0.9% spare agarose C1- to 200mm scale
4B reaches about 10mm long scale, set blood sedimentation tube it is cooling after, agarose becomes semisolid, can prevent blood sedimentation tube inner cell flow out but
The substance of the water and chemical analysis etc that will not prevent small molecule passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample database are protected
5 blood plasma of AIDS (AIDS) patient deposited, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and injects blood sedimentation tube (letter in batches
Easy purification device) upper end blank pipe, wait flow through the hybridoma macrophage strain layer of blood sedimentation tube lower layer and out of blood sedimentation tube after outflow, receipts
Collect efflux, blood plasma after referred to as AIDS filter.Blood plasma and blood plasma after filter before taking AIDS to filter, with human macrophage migration inhibitory factor
(MIF) ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) pairing detection, by specification operation, detection range
, can be under white background for 0~800pg/ml, susceptibility 1.0pg/ml, directly detect by an unaided eye: color is got in reacting hole
It is deep, it is positive stronger, negative reaction be it is colourless or extremely shallow, according to the depth of be in color, with "+", "-number indicate.OD can also be surveyed
Value: on ELISA detector, at 450nm (if developing the color with ABTS, 410nm), each hole OD is surveyed after returning to zero with blank control wells
Value, it is as positive if more than 2.1 times of defined negative control OD value.As a result such as table 1, MIF testing result is equal in blood plasma before filtering
For negative (or because content causes degradation etc. lower than detection sensitivity, blood plasma long-term preservation), and testing result is in blood plasma after filtering
It is positive.Illustrate that macrophage hybridoma cell strain produces MIF cell factor in this process.MIF be collection cell factor, growth because
Son, the multi-effect protein molecular of hormone and enzyme characteristic, as in inherent immunity and the performance of the regulatory factor of inflammatory reaction
The effect of pivot plays panimmunity function in various infection and active chronic inflammation disease.Making the filtration letter of AIDS blood plasma
Before and after easy clarifier while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 antigen
Detection kit (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known concentration 0pg/ml, 0.5pg/
The p24 antigen of ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are as control, minimum detection limit
Lower than 5pg/ml, 0~400pg/ml of measurement range, 450nm measures extinction in the range of linearity 0.5pg/ml~80pg/ml, 15min
It spends (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ml extinction not higher than 0.050,0pg absorbance value
Degree is not less than 1.000, is considered as the positive as absorbance > 0.12, and as a result (table 2) illustrates the simple purification of AIDS blood plasma filtration
After device, part HIV is swallowed by macrophage hybridoma cell strain to be adsorbed, and the blood plasma HIV after filtration is significantly reduced, through the 1st
After secondary filtration, HIV clearance rate is 20.55%, and after the 2nd filtration, HIV clearance rate is 42.83%, p < 0.01, is had obvious
Effect, illustrate with filtration number increase HIV can constantly be removed, thus reach treatment AIDS purpose.
1 AIDS blood plasma of table filter MIF testing result before and after the simple purification device of the macrophage containing hybridoma (it is quantitative:
pg/ml)
2 AIDS blood plasma of table filters the simple purification device front and back p24 testing result (p24:pg/ of the macrophage containing hybridoma
ml)
In short, above-mentioned simple confirmatory experiment shows easily to be fused into the more of large volume by the peripheral white blood cells of HIV infection
Core giant cell or many cells condensate can be separated by blood separator and be removed；And the HIV to dissociate in blood plasma, it can be by plasma purification
Agent (strain of hybridoma macrophage) is removed, and shows the AIDS plasma purification constituted with separator and clarifier (agent) for critical component
Therapeutic equipment has the significant therapeutic efficiency for removing the inside and outside inhibition of HIV of haemocyte.
Claims (10)
1. a kind of AIDS plasma purification therapeutic equipment for medical domain, which is characterized in that separated by blood separator, blood plasma
The external HIV purification device that device, the plasma purification device of exit setting sieve and circulation line are constituted, the plasma purification device
Filling contains the cells frozen storing liquid of 80% hybridoma macrophage strain concentration, and the hybridoma macrophage strain refers to by having phagocytosis
The macrophage of HIV function is merged with the myeloma cell with Immortalization performance and is made thin with the fusion for having the two performance
Born of the same parents, the external HIV purification device are used to filter out the HIV in multinucleate giant cell and blood plasma containing HIV.
2. AIDS plasma purification therapeutic equipment according to claim 1, which is characterized in that the blood separator by inner cavity and
Exocoel is constituted, and inner cavity and exocoel are communicated by the micropore on tube wall, and the aperture of the micropore is 1~250 μm, can pass through single blood
Liquid cell but large volume cell made of mutually cannot bonding or merge by two or more cell.
3. AIDS plasma purification therapeutic equipment according to claim 2, which is characterized in that the micropore hole of the blood separator
Diameter is 50~250 μm, 3~40 μm or 1~2 μm.
4. AIDS plasma purification therapeutic equipment according to claim 1 to 3, which is characterized in that selecting micropore size is 50
~250 μm of separator separates the fungi, spirillum and/or tumour cell of especially big volume；Selecting micropore size is 3~40 μm
Mononuclear macrophage, multinucleate giant cell, many cells condensate, the particulate matter for being adsorbed with HIV of separator separation HIV infection
And/or the CD4+ cell vulnerable to HIV infection.
5. AIDS plasma purification therapeutic equipment according to claim 1, which is characterized in that the plasma separator hollow fibre
Film diameter is 270~370 μm, and film thickness is 50 μm, and aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm, can be stopped
All cell filtrations.
6. AIDS plasma purification therapeutic equipment according to claim 1, which is characterized in that the clarifier is by its exit
Sieve and hybridoma macrophage strain collectively form molecular sieve mechanical removal and cellular immunity removes the barrier of blood plasma HIV.
7. AIDS plasma purification therapeutic equipment according to claim 6, which is characterized in that the volume of the clarifier is 200
~300ml, entrance top diameter cell screen clothes mesh number are 800 mesh, and 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, and setting promotes system between liquid entrance and cell screen clothes
System stablizes the buffer area of circulation.
8. AIDS plasma purification therapeutic equipment according to claim 1, which is characterized in that the cells frozen storing liquid is containing 30%
The 1640 culture medium of fetal calf serum and 12% dimethyl sulfoxide.
9. a kind of preparation of the AIDS plasma purification therapeutic equipment cleanser for medical domain, which is characterized in that hybridoma is huge
Phagocyte strain assembles the cylindrical cleaner made of acrylate high-biocompatibility material after cleaning with sterile saline,
Then plus cells frozen storing liquid to 4/5, make hybridoma macrophage strain concentration up to 80%, gently shake up, seal, through 4 DEG C of 0.5h ,-
Overnight, it is spare then to enter -196 DEG C of liquid nitrogen cryopreservations for 20 DEG C of 2h and -70 DEG C, thaws in use, needing rapidly throw cylindrical cleaner
Enter into 37 DEG C of water-baths having been warmed up, and constantly shake, melts the liquid in pipe rapidly, it is then clear with sterile saline
It is used after washing.
10. any AIDS plasma purification therapeutic equipment of claim 1-8 is preparing the application in purging in vitro device, special
Sign is, the connection structure of the purging in vitro device are as follows: one end of arteries and veins blood vessel (1) through heparin and blood pump (2) with contain
The blood separator (3) of waste liquid outlet (5) is connected, and blood separator (3) exports (4), blood pump (6), circulation line through blood
(7) be connected with plasma separator (8), the plasma outlet port of plasma separator (8) through blood plasma pump (9) and blood vessel (10) with two simultaneously
The clarifier (11,12) of connection is connected, the export pipeline (13) of two clarifiers and the haemocyte export pipeline of plasma separator (8)
(14) converge after converging through venous line (15).
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| CN201610540910.8A CN106110425B (en) | 2016-07-01 | 2016-07-01 | AIDS plasma purification therapeutic equipment |
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| CN201610540910.8A CN106110425B (en) | 2016-07-01 | 2016-07-01 | AIDS plasma purification therapeutic equipment |
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| CN106110425B true CN106110425B (en) | 2018-12-04 |
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| CN102631891A (en) * | 2012-04-23 | 2012-08-15 | 武汉大学 | Human immunodeficiency virus affinity adsorption column, and preparation method and uses thereof |
| CN103170023A (en) * | 2011-12-22 | 2013-06-26 | 张涛 | AIDS therapeutic equipment and manufacturing method |
| CN103691016A (en) * | 2014-01-15 | 2014-04-02 | 倪自谦 | Aids virus specific plasma adsorption column and application method thereof |
| CN104801278A (en) * | 2015-03-29 | 2015-07-29 | 武汉瑞法医疗器械有限公司 | Preparation method of AIDS (acquired immune deficiency syndrome) virus affinity adsorbent |
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| US20090081176A1 (en) * | 2007-09-07 | 2009-03-26 | Rudolph Maravich | Cure for the human immunodeficiency virus |
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| CN103170023A (en) * | 2011-12-22 | 2013-06-26 | 张涛 | AIDS therapeutic equipment and manufacturing method |
| CN102631891A (en) * | 2012-04-23 | 2012-08-15 | 武汉大学 | Human immunodeficiency virus affinity adsorption column, and preparation method and uses thereof |
| CN103691016A (en) * | 2014-01-15 | 2014-04-02 | 倪自谦 | Aids virus specific plasma adsorption column and application method thereof |
| CN104801278A (en) * | 2015-03-29 | 2015-07-29 | 武汉瑞法医疗器械有限公司 | Preparation method of AIDS (acquired immune deficiency syndrome) virus affinity adsorbent |
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