CN109157690A - Monkey-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain - Google Patents

Abstract

A kind of monkey for medical domain-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, it is characterized in that, using hematopoietin as inducer, cultivate positive " O " type rhesus macaque bone marrow cell of Rh, to increase the erythroid cells of orientation development, the rhesus macaque bone marrow cell after culture is followed by fused to hybrid cell with human myeloma cell, again with the differentiation of hematopoietin inducement crossbreeding cell directional, with HAT screening hybrid cell clone, the Rh positive cell hybrid strain that can be infinitely expanded in vitro with Rh antibody screening, adsorbent is used as after industrialization expands, absorber is made, then vitro Adsorption device is collectively formed with blood separator, by adsorbing the pathogenic Rh antibody in female tire blood group incompatibility maternal blood slurry, creation do not remove blood plasma and its beneficial without the honest and clean of plasma exchange liquid The treatment new method of female tire blood group incompatibility Hemolysis of valence safety.

Description

Monkey-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain

Technical field

The present invention relates to the preparations of medical domain mother's tire blood group incompatibility treatment hybrid strain, are mainly used for female tire Rh blood group incompatibility The absorption of anti-fetal red blood cells antibody is removed in pregnant woman blood plasma.

Background technique

The mankind have 26 kinds of blood groups such as ABO, Rh, MN, Lew and Kell, most important of which is that abo blood group and Rh blood group, ABO Staphylococal Protein A that blood group is carried according to red blood cell, B antigen, AB antigen are divided into A type, Type B, AB type and O-shaped without A and B antigen；Rh blood Whether type according to red blood cell carries D antigen, and to be divided into the Rh positive and Rh negative, containing D antigen on the red blood cell of Rh positive, can with it is anti- Immune agglutination occurs for D serum.The frequency of Rh feminine gender is about 0.4% in China's Chinese Han Population, and ethnic group of the Northwest with And the frequency of certain country's Rh feminine genders is up to 10% or more, so the incidence in these areas and country's Rh blood group incompatibility Hemolysis Compared with China's Chinese Han Population height.

Blood group antigens heredity can carry the blood group antigens from father, show as from parents, fetal red blood cells Different from the blood group of parent, at this time when the red blood cell of fetus enters the blood circulation of parent, parent can be induced and generate antibody, into And entered in fetus body by placenta, in conjunction with and destroy fetal red blood cells, lead to fetus and/or neonatal Hemolysis.Wherein The incidence that female tire abo blood group does not conform to is higher, but the incidence of fetus Hemolysis is lower, even if generation is also relatively light, seldom causes Nuclear icterus and oedema, the gestational period are not necessarily to specially treated.And Rh blood group incompatibility is positive since Rh feminine gender parent generates largely anti-Rh The Rh antibody (the anti-D of IgG) of fetal red blood cells, into after fetal circulation, the anti-D of IgG and Rh positive babies erythrocyte surface D antigen occur it is immune combine, cause a large amount of fetal red blood cells to be destroyed, make fetus severe anemia, anoxic, heart failure, liver Damage, Hypoproteinemia, anasarca, hydrothorax, ascites even Intrauterine Fetal Death.In non-neonate, Rh blood group incompatibility haemolysis goes out The time of existing jaundice does not conform to haemolysis person's morning compared with abo blood group, can occur in 12 hours after birth earliest, and majority goes out in 24 hours It is existing.Since a large amount of bilirubin that haemolysis generates cannot exclude in time, aggravate icterus neonatorum, it is thin that a large amount of bilirubin penetrates into brain Born of the same parents and cause nuclear icterus, often die of severe anemia, heart failure, nuclear icterus, the death rate is very high.

Although certain pregnant woman can prevent the generation of female tire Rh blood group incompatibility Hemolysis, mesh by injecting Rh antibody in advance Preceding most countries have been forbidden to use D antigen and volunteer are immunized, therefore the source of polyclonal Rh antibody is extremely limited, and by EB Virus Transformation lymphocyte first converts the Rh antibody for hybridizing preparation afterwards in vivo, there are still Epstein-Barr virus, HIV and hepatitis virus Etc. pathogen potential pathogenic problem, and the Rh antibody prepared with hybridoma technology is because having mouse oncogene or containing source of mouse Albumen and easily cause allergic reaction, thus make by internal injection Rh antibody prevent Rh Hemolysis method be restricted.

Clinically mainly there are pregnancy period plasma exchange, fetal transfusion, end to the treatment of female tire Rh blood group incompatibility Hemolysis at present Only gestation and neonatal exchange transfusion treatment.Blood exchanging therapy or blue light illumination, note are mainly such as used to the newborn of hyperbilirubinemia Penetrate immunoglobulin；The gestational period, anti-D potency need to consider intrauterine transfusion, including fetus peritoneal transfusion or blood at 1: 32 or more Blood transfusion in pipe, both of which has certain risk and none is proved effectively；The gestational period, anti-D potency needed to consider at 1: 64 or more Replacement therapy of blood plasma, i.e., in vitro in circulation path with filter method separation pregnant woman haemocyte and blood plasma, remove blood plasma and with etc. (each replacement amount is 2 000~3 000ml, speed as displacement liquid supplement feedback for the fresh frozen plasma of amount or 5% albumin It is 2~4h for 20~30ml/min, treatment time, every 1~3d is treated 1 time).Or take pregnant woman's whole blood about 400ml every time, low temperature from Its blood plasma about 300ml is removed after the heart, supplements equivalent homotype fresh plasma, also defeated Autoerythrocyte.Plasma exchange can be removed Plasma volume proportionally reduces the titre (potency) of pathogenic antibody, so as to extend fetus in female intracorporal survival and development Between, it is that female tire ABO, Rh or other blood group incompatibility pregnant woman prevent miscarriage the good selection for the treatment of clinical early stage, without apparent adverse reaction, Tool has a better effect.But plasma exchange can only remove the partial antibody in blood, cannot terminate antibody and continue to generate, also not Female tire blood group incompatibility Hemolysis for having occurred can be reversed, it is just effective to need to carry out continual plasma exchange, is only applicable to once exist Fetus edema occurred before 20~22 weeks for gestation or spouse is the pregnant woman of homozygote pathogenic antigens, especially caused a disease in removal part While antibody, a large amount of blood plasma for having also been removed the composition containing multiple beneficial can not be mended completely although being supplemented with displacement liquid The blood plasma and its beneficial being removed enough, and the displacement liquid dosage substituted is big, and somewhat expensive, allosome blood plasma easily causes infectious disease With infusion reaction etc., which also limits the generally developments of plasma exchange.

It has been reported that can be using positive " O " the type human red blood cells of Rh or rhesus monkey erythrocytes, after specially treated, system It is assembled into extracorporeal circulation apparatus at Rh positive red blood cell absorber (column), and then with blood separator, for removing in pregnant woman's body Rh antibody, i.e., the peripheral blood of pregnant woman is drawn into extracorporeal circulation apparatus, blood plasma and haemocyte, blood plasma is isolated with blood separator In Rh antibody pass through Rh positive red blood cell absorber (column) when, Rh antibody therein is adsorbed by Rh positive red blood cell, adsorb Rh After blood plasma outflow absorber (column) after antibody, converge with the haemocyte separated before, then feeds back in vivo, reach treatment mesh 's.Theoretically see that this treatment method is substantially better than existing treatment method, but the problem is that, these documents are not yet It is proposed the amplification in vitro method of positive " O " the type red blood cell of Rh, positive " O " the type human red blood cells of Rh used or rhesus monkey erythrocytes are only Human body or the rhesus macaque of the Rh positive can be directly derived from.Because without establish in vitro infinitely positive " O " the type human red blood cells of amplification Rh or The method of rhesus monkey erythrocytes relies on so these treatment methods reported in the literature have positive " O " the type people of Rh or rhesus macaque Property, it is difficult to reach the industrial application for being detached from organism, and the risk with the input of foreign body blood source.

It is another it has been reported that gram strangle (Kohler) and Millstein (Milstein) proves in 1975, myeloma cell and Immune animal splenocyte hybridization, forms antibody with high specificity -- the monoclonal antibody that can be secreted for corresponding antigens, this skill Art is commonly referred to as hybridoma technology.Myeloma cell can continuous passage in vitro, and splenocyte cannot be bred in vitro.If will The myeloma cell of mouse hybridizes with the lymphocyte that can secrete certain antibody or the factor, then hybrid cell both has tumour cell The characteristic of Immortalization, and the ability with lymphocytic emiocytosis specific antibody or the factor, while also overcoming immunoblastic lymphoma Cell cannot in vitro Immortalization the shortcomings that, the cell of this hybridization is known as hybridoma.

It is reported according to document above, applicant is thin with positive " O " the type human red blood cells of Rh and the rat bone marrow tumour of energy indeterminate growth Born of the same parents are merged, it is intended to which preparation not only with the antigenic of positive " O " the type human red blood cells of Rh but also had myeloma cell's indeterminate growth The fused cell of characteristic, to expand positive " O " the type human red blood cells (composition) of Rh by the unlimited of fused cell, to establish People-mouse cell fusion mother's tire blood group incompatibility treatment hybrid strain preparation method, but do not succeed always.Followed by attempt to distinguish again Immortality is prepared in the method for Epstein-Barr virus, SV40LT (simian virus SV40 large T antigen), hTERT (reverse transcriptase of telomere) transfection Change bone marrow cell strain, and attempts to cultivate, filters out Rh positive " O " type human red blood cells system, it is red thin to establish positive " O " the type people of Rh The amplification in vitro method of born of the same parents also fails to succeed always.Applicant is intended to find or manufacture experimently out that the non-of Rh antibody can be adsorbed Red blood cell class material, also has no resolution so far.

In order to realize that only removal pathogenic antibody is without removing blood plasma and its cheap of multiple beneficial composition, safety and without pair The treatment of female tire blood group incompatibility Hemolysis of effect, and in order to save blood source, it is necessary to create a kind of positive " O " type red blood cell of Rh The unlimited amplification method of (composition) with positive " O " the type red blood cell (composition) of industrialization preparation Rh, and then is used for Rh as adsorbent It is prepared by the industrialization of antibody absorber (column).

Summary of the invention

In order to solve the problems, such as the treatment of the female tire blood group incompatibility Hemolysis troubled for a long time, present inventors have proposed the present invention.

The invention aims to provide the preparation method of female tire blood group incompatibility treatment hybrid strain, another object is to provide for Made hybrid strain is treating the application in female tire blood group incompatibility Hemolysis.

The object of the present invention is achieved like this: using hematopoietin as inducer, rhesus macaque bone marrow cell is cultivated, To increase the erythroid cells of orientation development, followed by the rhesus macaque bone marrow cell through cultivating is configured to mix with human myeloma cell Cell, the effect through polyethylene glycol are fused to hybrid cell, then with the directed differentiation of hematopoietin inducement crossbreeding cell, With HAT screening hybrid cell clone, the rhesus monkey erythrocytes hybrid strain that can infinitely expand in vitro, warp are screened with Rh antibody reagent It further is used as adsorbent after amplification, absorber is made, for adsorbing the pathogenic antibody of female tire blood group incompatibility Hemolysis.

More specific says, is contained with the bone marrow cell cultures culture of the hematopoietin Han 3.1~4.1IU/mL more Kind of candidate stem cell but the rhesus macaque bone marrow cell that can not infinitely pass in vitro for 24 hours~48h, then by the rhesus macaque through cultivating Bone marrow cell is mixed with the human myeloma cell (sp2/0) that can infinitely pass in vitro with 2: 1, in 30% polyethylene glycol fusion agent (PEG1500) under the action of, make rhesus macaque bone marrow cell and human myeloma cell is fused to have both two Cell Components and characteristic Hybrid cell is followed by the DMEM culture medium containing 2.5~3.1IU/mL hematopoietin, 2 × HAT and 15% fetal calf serum Then selection culture picks them separately each clone cell and carries out sub-bottle expansion culture or in 6 orifice plates to screen hybrid cell clone Carry out point hole and expand culture, finally take suitable amplifying cells from each clone respectively, respectively with Rh antibody (containing anti-D) blood It mixes clearly, occurring agglutinating particle in 3~5 minutes, person is the made rhesus monkey erythrocytes hybridization that can infinitely expand in vitro Strain, and then as adsorbent, it is used to prepare Rh antibody absorber, then collectively forms extracorporeal circulation apparatus with blood separator, By adsorbing the Rh antibody in female tire blood group incompatibility maternal blood slurry, reach therapeutic purposes.

The present invention according to have been widely used but be limited only to monoclonal antibody preparation mouse immunized B cells and rat bone marrow tumour it is thin The hybridoma technology of born of the same parents' fusion, devises the fusion method of Rh positive Rhesus bone marrow cell and human myeloma cell, in tradition Hematopoietin is creatively referred on the basis of hybridoma technology, is increased using hematopoietin as inducer Bone marrow cell merge before incubation step, to promote the directed differentiation and increment of red system's early stage cell, to increase melting for red system Conjunction rate.And hematopoietin takes part in the complete of bone marrow cell fusion, hybrid cell screening and its cloning passage amplification Journey induction, be conducive to it is red be each phase cell and D antigen differentiation and maturation, thus prepare the rhesus macaque that can infinitely expand in vitro Red blood cell hybrid strain is used as adsorbent after industrialization expands, absorber is made, then collectively forms with blood separator in vitro Circulator, by adsorbing the pathogenic Rh antibody in female tire blood group incompatibility maternal blood slurry, creation do not remove blood plasma and its The treatment new method of female tire blood group incompatibility Hemolysis of the inexpensive safety without plasma exchange liquid of beneficial.

Specific embodiment

Fig. 1 is the made hybrid cell clone figure of the present invention.

Fig. 2 is the immune agglutination figure of the made hybrid cell and corresponding antibodies of the present invention.

Fig. 3 is the application schematic diagram of female tire blood group incompatibility treatment hybrid cell strain proposed by the present invention.

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

Fig. 5 is the schematic diagram of internal structure of the absorber proposed according to the present invention

In Fig. 1, the immature erythrocyte system that rhesus macaque bone marrow cell breaks up through hematopoietin directional induction, Under the action of PEG, after being merged with human myeloma cell, is screened 1~2 week through HAT, shoot (40X) under inverted microscope, obtain It clones and schemes to hybrid cell.

In Fig. 2, to hybrid cell clone as shown in Figure 1 obtained makees further screening and culturing, cloning passes Then generation and hematopoietin directional induction take the hybrid cell for being considered red blood cell D antigen differentiation and maturation, are made into Cell suspension mixes in proportion with anti-D (IgM+IgG) blood grouping reagent, through slide method agglutination test, obtains antigen and antibody The graininess immune agglutination figure of immune association reaction result, if the slide left side (1) in Fig. 2 indicates agglutinating particle, the right (2) are Negative control.

In Fig. 3, 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) absorption in parallel with two through blood plasma pump (6) and circulation line (7) Device (8,9) is connected, and is then successively connected with circulation line (10), venous line (5), the other end and vein of venous line (5) Blood vessel is connected.

In Fig. 4,1 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. 5,1 is absorber, and 2 be the positive red system's hybrid strain of Rh in absorber, 3 be into absorber Rh it is anti- Body, 4, which be Rh antibody, is combined by the positive red system's hybrid strain of Rh and is formed by antigen antibody complex, and 5 be RBC fragment.

Below with reference to Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Fig. 5, embodiment of the present invention is explained in detail.

1, cell to be fused

(1) bone marrow cell: rhesus macaque bone marrow cell is purchased from zoopery mechanism.

(2) myeloma cell: human myeloma cell (H929) is purchased from Sigma company.

2, experiment reagent

(1) hematopoietin: it is purchased from Sigma company.

(2) DMEM culture medium, HAT Selective agar medium and PEG are purchased from Sigma company；Fetal calf serum (FBS) is purchased from Gibco Company；DMSO (dimethyl sulfoxide) is domestic analytical reagents.

(3) Rh antibody serum (the anti-D of IgG): it is purchased from Sigma company.

3, cell fusion method

(1) it the hematopoietin Fiber differentiation before bone marrow cell fusion: is incorporated in traditional bone marrow cell cultures 3.1~4.1IU/mL hematopoietin gently shakes up after being inoculated with bone marrow cell, sets 37 DEG C of culture for 24 hours~48h.Design The purpose of the step is to promote bone marrow cell to break up to red system's early stage cell directional using hematopoietin as inducer, with Increase the cell quantity of red system, to improve the fusion rate of erythroid cells.

(2) preparation before bone marrow cell fusion: the marrow through 3.1~4.1IU/mL hematopoietin Fiber differentiation is thin Born of the same parents adjust total cell number to 1 × 10 with DMEM culture solution (basal medium)⁸~2 × 10⁸, blue dyeing, phase contrast microscope are expected with platform Check that viable count, viable count are higher than 80% for qualification.

(3) preparation of myeloma cell: fusion the last week takes out the myeloma cell (SP2/0) frozen out of liquid nitrogen container, It is immediately placed in hot water defrosting.Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min；It is repeated 1 times.Sediment is moved Enter in Tissue Culture Flask, add DMEM culture solution, set CO2 incubator culture, once passed on or expanded culture within 3~4 days, merges Cell state is adjusted in first 24 hours, guarantees that cellular morphology is good before merging, growth is vigorous.By human myeloma cell when fusion (H929) it blows down, is transferred in centrifuge tube from culture bottle, 1000r/m is centrifuged 5~10min, washes repeatedly cell 2 times, gently strikes It beats and mixes, take a small amount of suspension to count, adjust density, make the density 80% or so when fusion

(4) cell fusion: gently rotation preheats centrifuge tube in 37 DEG C of water-baths, aseptically by preheating after taking-up The 30%PEG3000 of 1000 μ L is added drop-wise in fusion pipe in 60s along tube wall, while gently rotating centrifugal pipe, later will preheating 25mL basal medium be also added drop-wise in centrifuge tube along tube wall in 3~5min, lightly rotated during addition from Heart pipe, is then allowed to stand in 37 DEG C of water-bath 10min, is centrifuged 5min with 1500r/m, discards supernatant, 50mL HAT culture medium is added, fits It is inoculated into after mixing in 96 well culture plates, is placed in 37 DEG C, cultivates in 5%CO2 incubator.

4, the screening of fused cell

The cell growth status in 96 orifice plates is observed, is only that the cell of effective integration can be grown after 7~10 days, abandons at this time HAT culture medium is removed, the complete medium containing HT is replaced, continues to cultivate, hybrid cell clone's (figure of round light can be observed 1)。

5, the monoclonal screening of hybrid cell and colonized culture

(1) monoclonal screening (subclone method): when fused cell clonal growth area reaches 1/10 cell hole, removal Culture solution selects the good culture hole of hybrid cell growth conditions, marks cell growth position, size under microscope, use nothing Cell clone is drawn in the new culture hole for having complete medium by bacterium pipette tips in the position of mark, then successively doubling dilution It arrives and counts hole below, make cell number 0~1 in each hole below, 37 DEG C, 5%CO2 incubator is interior to be cultivated about 1 week, microscope Lower observation cell growth status, when the cell clone when below in (inverse) hole is covered with to 1/10 or more hole floor space, selection is ( Number) row monoclonal screens again for cell in the hole of back, prepare monoclonal hybrid cell.

(2) colonized culture: the monoclonal hybrid cell through subclone screening is subjected to secondary culture, expands hybrid cell Increase to required cell quantity, for screening Rh positive Rhesus red blood cell hybrid strain (female tire blood group incompatibility treats hybrid strain).

6, the screening of female tire blood group incompatibility treatment hybrid strain (the Rh positive " O " type rhesus monkey erythrocytes hybrid strain)

(1) screening of " O " type red blood cell hybrid strain: the monoclonal hybrid cell for colonized culture of learning from else's experience, routinely " ABO " The identification method of blood group is mixed with anti-A, anti-B serum respectively, and it is " O " type red blood cell hybrid strain that agglutination person does not occur both.

(2) screening of positive " O " type red blood cell hybrid strain of Rh: " O " type red blood cell hybrid strain for above-mentioned screening of learning from else's experience, respectively It is mixed with Rh antibody (containing anti-D) serum, routinely agglutination person's (slide method, occurs in 3~5 minutes in the identification method of Rh blood group Agglutination test: Fig. 2) it is positive " O " type rhesus monkey erythrocytes hybrid strain (the female tire blood of the made Rh that can infinitely expand in vitro Type does not conform to treatment hybrid strain).

(3) female tire blood group incompatibility treats freezing for hybrid strain: the hybrid strain of above-mentioned preparation, by the conventional cryopreservation side of cell strain Method freezes in -196 DEG C of liquid nitrogen, is ready for use on industrialization preparation.

7, the industrialization preparation of female tire blood group incompatibility treatment hybrid strain

(1) the industrialization amplification of female tire blood group incompatibility treatment hybrid strain: prepared female tire blood group incompatibility treats hybrid strain With the performance infinitely expanded, it can be used for industrialization secondary culture and amplification.

(2) purification of female tire blood group incompatibility treatment hybrid strain: the female tire blood group incompatibility treatment hybrid strain for taking industrialization to prepare, It is miscellaneous with isometric physiological saline cleaning with the centrifugal speed of 1500r/min × 5min respectively under conditions of 4 DEG C and 37 DEG C It hands over strain 4~5 times, to remove the antibody and harmful substance that hybrid strain surface may be adhered to.

(3) removal of female tire blood group incompatibility treatment hybrid strain hemoglobin: with 0.04mol/L Na₂HPO₄And 0.04mol/L NaH₂PO₄The PB lysate that osmotic concentration is respectively the PH7.4 of 25mmol/L and 35mmol/L is prepared, in 4 DEG C and 2500r/min Under the centrifugal condition of × 10min, alternately and repeatedly Washed Red Blood Cells, are changed by the alternating of osmotic concentration, remove the blood of hybrid strain Lactoferrin is finally cleaned with physiological saline colourless to supernatant.

(4) the antigenicity test of female tire blood group incompatibility treatment hybrid strain: hybrid strain D antigen 1. is detected with anti-D standard items: By made hybrid strain and anti-D standard items in 37 DEG C of incubation 5min, after centrifugation, supernatant is taken to detect the potency of anti-D standard items, root Numerical value is reduced according to potency to determine the antigenicity of hybrid strain.Specific method is: taking 10, test tube, respectively number 1~10, the 1st pipe 40% hybrid strain precipitating 1.0ml is added, then other each pipes plus physiological saline 0.5ml draw hybrid strain precipitating from the 1st pipe 0.5ml is added to the 2nd pipe, and 0.5ml to the 3rd is inhaled after mixing and is managed, and is diluted to the 10th pipe with same operation, finally 0.5ml is sucked out in pipe It discards, every pipe adds anti-D standard items 0.5ml, 37 DEG C of incubation 5min to mix gently after centrifugation, dilute with the maximum for occurring being aggregated completely The antigenicity that multiple represents hybrid strain is released, extension rate is bigger, and antigenicity is stronger, and the ability for adsorbing Rh antibody is stronger.2. using Rh (D) normal erythrocytes measure supernatant antibody titer, and it is as miscellaneous that anti-D standard items potency (known) subtracts supernatant antibody titer Hand over the antigenicity (potency) of strain.Supernatant antibody titer detection method are as follows: take 10 test tubes, respectively number 1~10, each pipe is added Physiological saline 1ml takes supernatant 1ml to be added to removal 1ml after the 1st pipe mixes and goes to the 2nd pipe, is diluted to the 10th with same operation Pipe, finally pipe is sucked out 1ml liquid and discards, and the 40 μ l of serum diluted is mixed with 10 μ l of Rh (D) normal erythrocytes, is incubated for 10 points Clock is centrifuged 5 minutes interpretation results, and the agglutination pipe of maximum dilution multiple is antigenic intensity (potency), and potency should be 1: 1500 More than.

(5) the content of hemoglobin test of hybrid strain: with the methods of hypotonic destruction hybrid strain, with conventional chemical luminesceence analysis Method or fluorescence analysis, the qualitative content of hemoglobin in quantitative detection hybrid strain should be lower than the reference value of human normal plasma Lower bound.

(6) the storage liquid of female tire blood group incompatibility treatment hybrid strain and storage method: 3.5% mannitol saves liquid: taking sweet dew Alcohol 17.5g, citric acid trisodium 1.5g, citric acid 0.2g, glucose 7.93g, sodium dihydrogen phosphate 0.94g, adenine 0.14g, chlorine Change sodium 4.97g, is dissolved in the distilled water of 500ml.Above-mentioned female tire blood group incompatibility through antigenic test passes is treated into hybrid strain It is configured to 80% positive " O " type rhesus monkey erythrocytes hybrid strain of Rh, 4 DEG C of storages are spare.

8, female tire blood group incompatibility treatment hybrid strain is used to prepare absorber

(1) preparation principle: female tire Rh blood group incompatibility Hemolysis is because the free Rh antibody in pregnant woman blood plasma enters fetus In vivo, fetal red blood cells are made to be destroyed and cause a disease.But Rh antibody can be adsorbed by corresponding Rh positive red blood cell.

(2) absorber prepares material: select without complement activation, no inflammation reaction, without leucocyte, blood platelet, blood oxygen pressure, The high-biocompatibility material close to Human vascular endothelial that complement C 3 C5a changes can pass through the sides such as covalent, grafting, polymerization Method improves uniformity, the hydrophily of material surface, reduces the influence to blood coagulation and oxidative stress.Add in absorber inner surface hydrophilic Biocompatibility can be improved in gel.Certain anticoagulant substances are solidificated in absorber inner surface, blood clotting is can inhibit, reduces Heparin dosage even realizes no-rod tractor, and such as heparin is aggregated on polyacrylonitrile-polyethyleneimine film, can reduce allergic constitution Allergic reaction；Heparin covalent is integrated to polyether sulfone surface, the mechanical property of polyether sulfone can be kept and improves table in absorber The anticoagulation function in face.The covalent immobilisation linoleic acid film on cellulose acetate film, or the linoleic acid that polyacrylic acid will be covalently bound to It is grafted onto polysulfones film surface, can there is preferable histocompatbility and anticoagulant effect.

(3) adsorber enclosure specification: the hydrostatic column that the bottom diameter for being made 50mm × 60mm is small, top diameter is big, or the side of being made Shape, infundibulate, volume about 200~300ml, entrance are equipped with buffer area and top diameter cell screen clothes, and top diameter cell screen clothes mesh number is 500 mesh, exit are equipped with bottom diameter cell screen clothes and cell strainer, and bottom diameter cell screen clothes mesh number is 50 mesh, and cell strainer mesh number is 200 mesh are also equipped with buffer area, are conducive to the stability of system circulation between bottom diameter cell screen clothes and cell strainer.

(4) filling of absorber: taking female tire blood group incompatibility treatment hybrid strain to be packed into absorber, until 4/5, it is sweet to be added 3.5% Reveal alcohol and save liquid, make hybrid strain concentration up to 80%, jog mixes, and capping is set 4 DEG C and saved backup.Repeat to be centrifuged before use It goes mannitol to save liquid and supplies the operation of hybrid strain cell, make hybrid strain cell tight full of absorber.

9, absorber constitutes female tire blood group incompatibility pathogenic antibody adsorbing therapy device

Adsorbing therapy device of the invention is the extracorporal circulatory system dress being made of absorber, plasma separator and other attachmentes It sets.

(1) absorber: preparation method is as described above, the internal filling female tire blood group incompatibility treatment that can adsorb pathogenic antibody is miscellaneous Hand over strain.

(2) plasma separator (washed corpuscles and blood plasma): 1. preparation principle: according to the molecule of haemocyte and blood plasma components Size preparation, such as the size of visible component (haemocyte) in blood of human body are as follows: normocyte is about 7 microns (μm), is double Recessed discoid cell.Leucocyte is divided into 5 kinds, and about 12 μm of neutrophil leucocyte, eosinophil is more bigger, basophilic granulocyte Close with neutrophil leucocyte, 6-8 μm of small lymphocyte, 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 is differed, and the platelet mean diameter of people is 2-4 microns, 0.5~1.5 micron thick. 2. preparing material: poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. can be selected, 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 microinhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxidation Stress influence, the generation to improve sieving adequacy and biocompatibility, reduce complication.3. specification: with regard to separator Shape can be prepared into column construction as filter core with materials such as acetate fiber or absorbent cotton, make filter core with materials such as poly-vinegar non-woven fabrics It is prepared into the shapes such as flat structure.Aperture is determined by the molecular size of haemocyte and blood plasma components to be separated.Involved by the present invention And plasma separator with the high high molecular polymer of property stabilization, good biocompatibility, permeability be made hollow fibre type filter Device, hollow-fiber film diameter be 270~370 μm, film thickness be 50 μm, aperture be 0.2~0.6 μm, fibre length be 13.5~ 26μm.Blood plasma filtration is only permitted in the hole, but can stop all cell components.

(3) other attachmentes: 1. sound pulse pressure monitors: the arterial blood pressure monitoring mainly blocking to dynamic monitoring absorber micropore Situation, in addition to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When blood flow deficiency, angiosthenia will be reduced；When When having blood coagulation, thrombosis, especially absorber blockage of the micro orifice, angiosthenia will be increased.Vein pressure monitoring is used to monitor pipeline The pressure of blood backflow, when absorber blockage of the micro orifice, blood coagulation, thrombosis, blood flow is insufficient, venous return and syringe needle fall off When, 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 transducer potector, the general principle for using ultrasonic listening are Avoid patient that air embolism occurs and is arranged.When having monitored air bubble, detection system can drive artery and vein bloody path to press from both sides Carry out blocking blood flow, prevents dangerous generation.3. blood pump (Blood Pump): for pushing blood circulation, with the suitable of maintenance therapy Benefit carries out.Usual blood pump has rotary test speed function, and to monitor the blood flow of patient, therefore blood pump runner is wanted with the setting of groove spacing Accurately, and often adjustment is needed, the case where according to bloody path pump line road, spacing is generally set as 3.2~3.3mm, can not be too loose, Otherwise it is inaccurate to will cause blood flow detection；Also can not be too tight, it otherwise will cause pipe breakage.4. heparin pump (Heparin Pump): Heparin pump is equivalent to the micro-injection pump clinically applied, to continue the injecting heparin in sieving pipeline (patient blood), by The blood of patient Yu recycles contact with air in vitro, is easy to happen blood coagulation phenomenon, anticoagulative can be occurred using heparin pump. 5. additionally including temperature control, with parts such as liquid, degasification, monitored conductivity, ultrafiltration monitoring and leakage blood monitorings.

10, the application method of female tire blood group incompatibility pathogenic antibody adsorbing therapy device

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

(2) it is vented: with sterile saline filling liquid separator, absorber and each circulation line, excluding plasma separator, inhales Gas, bubble in adnexa and its circulating line, go through, and confirmation after gas, bubble without using.

(3) lead to liquid: arterial blood line pipe 1 is connected to the arteries of patient, whether go through exhaust again in operation Completely, 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: arterial blood line pipe (1) is connected to the arteries of curer, venous line (5) are connected curer's Then vein blood vessel opens blood pump, blood flow is 100~150ml/min, such as Fig. 3, when arterial blood is through arterial blood line pipe (1) when entering plasma separator (4), the blood plasma separated reaches first through circulation line (7) under the action of blood plasma pump (6) Absorber (8) begins releasing blood plasma wait be full of blood plasma, about 10 minutes, flows out through circulation line (10), synchronous to the second absorption Device (9) perfusion blood plasma starts again at perfusion blood plasma when the blood plasma in the first absorber (8) has nearly flowed, the second absorption at this time Device (9) begins releasing blood plasma, and two absorbers (8,9) in parallel are alternately.Such as Fig. 4, when blood to be separated enters blood plasma When inner cavity (2) of separator (1), the effect through valve (8) can be entered by the small molecule blood plasma and its composition (5) of micropore (3) Then the exocoel (6) of separator is flowed out through plasma outlet port (7), and cannot be flowed by the haemocyte (4) of micropore (3) through valve (8) Out.Such as Fig. 5, when Rh antibody (3) enters absorber (1) with blood plasma, the Rh positive hybrid strain (2) being adsorbed in device is combined into anti- Original antibody compound (4) and no longer move down, the cell fragment (5) that is destroyed and it is in combination made of macromolecular antigen it is anti- Nanocrystal composition cannot be by the mesh screen that absorber exports by detention, blood that blood plasma after absorption and plasma separator are separated It is fed back after cell confluency, so until the plasma circulation amount (usually 9L) being previously set, treatment just ends, if mating Computer program control, entire therapeutic process is controlled by computer, and can detect working condition at any time, using can it is more convenient, from Dynamicization and safety.

11, the creative use compliance test result of hematopoietin (EPO)

(1) hematopoietin (EPO) can improve red system's fusion rate: respectively with the tradition without hematopoietin Marrow medium and marrow medium of the present invention containing 3.1~4.1IU/mL hematopoietin under equal conditions carry out Bone marrow cell inoculation and culture and cell fusion, colony screening and red under equal conditions are carried out to the bone marrow cell after culture It is that (identification method is aforementioned referring to the present invention, has 1 pipe in 10 pipes and appears above 3~5 cell agglutination persons and sentences for D Antigen Identification Break as D antigen-positive cell), as a result, it has been found that, red system's fusion rate of the bone marrow cell through hematopoietin Fiber differentiation is bright It is aobvious to increase.As shown in table 1, as a result 3 bone marrow cell samples are generated respectively through methods experiment of the present invention with promoting erythrocyte The bone marrow cell of plain Fiber differentiation is found to have red blood cell D antigen through cell fusion, colony screening and red system D Antigen Identification Red system clone quantity is total up to 57, and not with the bone marrow cell of hematopoietin Fiber differentiation through cell fusion, clone Screening and red system D Antigen Identification, the red system clone's quantity for being found to have red blood cell D antigen are total up to 17, and the two has significantly Difference illustrates that hematopoietin can be in the directed differentiation of the red system of medullary microeirculation moderate stimulation, so as to improve red system Fusion rate.

Influence of hematopoietin (EPO) Fiber differentiation of 1 bone marrow cell of table to red system's fusion rate

(2) hematopoietin (EPO) can make D antigen differentiation and maturation: traditional bone to be free of hematopoietin 5 bone marrow cells of marrow culture medium culture, are divided into 2 parts for the bone marrow cell after culture respectively, respectively to contain 2.5~3.1IU/mL 15% fetal calf serum DMEM culture medium of hematopoietin and 15% fetal calf serum DMEM without hematopoietin Culture medium under equal conditions carries out cell fusion, colony screening and secondary culture, then carries out D Antigen Identification (identification method It is aforementioned referring to the present invention, there is 1 pipe in 10 pipes and appear above 3~5 cell agglutination persons and be judged as D antigen-positive cell, i.e., Rh is positive), clone's quantity of the Rh positive is confirmed, as a result such as table 2.As known from Table 2, melted with hematopoietin mediated cell Close, colony screening and secondary culture as a result, screen 59 Rh positive cell clones in total, and without hematopoietin Mediation person only screens 23 Rh positive cell clones, and the two has apparent difference, illustrates that hematopoietin can make D anti- Former differentiation and maturation.

Influence of 2 hematopoietin of table (EPO) to erythron D antigen directed differentiation

12, application effect of the invention verifying

In order to verify the present invention remove Rh antibody the effect of, devise easy test method: take sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 9, the made female tire blood group incompatibility treatment hybrid strain cell precipitation of the absorption present invention to 250mm scale, Followed by drawing suitable heat preservation after 100 DEG C dissolve in 42 DEG C of 1.0% spare agarose C1-4B, after agarose is cooling at For semisolid, hybrid strain can be fixed in pipe, easy garden cylindricality absorber is made with this.In addition fresh frozen plasma is bought 200mL and Rh antibody (the anti-D type serum dry powder standard items of people, Guangzhou Lian Tai Bioisystech Co., Ltd), is made into 1 with fresh plasma : 400,1: 400,1: 600 antibody titer, routinely Rh (anti-D) titre detection method (reference book), further detects confirmation Whether above-mentioned prepared antibody titer is consistent, (Rh) antibody (titre) before referred to as filtering, antibody difference before then respectively taking 10ml to filter The upper end blank pipe for injecting 3 blood sedimentation tubes collects efflux after flowing through blood sedimentation tube, (Rh) antibody after referred to as filtering, routinely Rh (anti-D) titre detection method confirms titre, then antibody after filter is passed through the erythrocyte sedimentation rate containing female tire blood group incompatibility treatment hybrid strain respectively It filters out for Guan Zhongzuo the 2nd time, is so repeated 3 times filtration and antibody titer detection, as a result (table 3) illustrate, and the filtration of Rh antibody is easily After absorber, most of Rh antibody is by corresponding female tire blood group incompatibility treatment hybrid strain absorption, through the 1st time, the 2nd time, the 3rd time After filtration, the average titer of Rh antibody respectively from 1: 467 before filter be reduced to filter after 1: 267,1: 167,1: 77, illustrate with filter The increase of number is crossed, Rh antibody can be removed constantly by the treatment hybrid strain absorption of female tire blood group incompatibility, so that reaching reduces pregnant woman (newborn) blood plasma Rh antibody titer and the purpose for treating female tire blood group incompatibility Hemolysis.

3 Rh antibody of table filters the absorber front and back titre testing result (l/x) of female tire blood group incompatibility treatment hybrid strain

Claims (9)

1. the preparation that a kind of monkey-people's cell merges female tire blood group incompatibility treatment hybrid strain, which is characterized in that merged in bone marrow cell The merging of preceding cell increment culture and the bone marrow cell after increment culture and human myeloma cell, hybrid cell screening, gram In Longhua culture and passage amplification, with the orientation division of the red system of erythropoietin stimulating, the differentiation and maturation of Rh antigen, it is made Positive " O " type rhesus monkey erythrocytes hybrid strain of the Rh that can infinitely expand in vitro, is used to prepare absorber, and then separate with blood plasma Device constitutes female tire blood group incompatibility pathogenic antibody adsorbing therapy device.

2. monkey according to claim 1-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In in the cell increment culture before bone marrow cell fusion, by hematopoietin by the dosage supplying of 3.1~4.1IU/mL Routine bone marrow cell culture medium.

3. monkey according to claim 1-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In, in cell fusion, hybrid cell screening, colonized culture and passage amplification, by hematopoietin by 2.5~ The dosage of 3.1IU/mL is incorporated the DMEM culture medium of 15% fetal calf serum.

4. monkey according to claim 1-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In the positive red system's hybrid strain of " O " type people of the Rh is with 0.04mol/L Na₂HPO₄With 0.04mol/L NaH₂PO₄The infiltration of preparation Concentration is respectively that the PB lysate of 25mmol/L and 35mmol/L alternately washs its hemoglobin contained.

5. monkey according to claim 1-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In positive " O " type rhesus macaque of 80%Rh that perfusion is made into inside the absorber with the cell-preservation liquid containing 3.5% mannitol is red The cell suspension of cell hydridization strain concentration, for adsorbing the Rh antibody flowed through in the blood plasma of absorber.

6. according to claim 1, monkey described in 5-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In the cell-preservation liquid of 3.5% mannitol is mannitol 17.5g, citric acid trisodium 1.5g, citric acid 0.2g, glucose 7.93g, sodium dihydrogen phosphate 0.94g, adenine 0.14g, sodium chloride 4.97g, are dissolved in the distilled water of 500ml.

7. according to claim 1, monkey described in 5-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In the shell volume of the absorber is 200~300ml, and entrance is equipped with buffer area and top diameter cell screen clothes, pushes up diameter cell sieve Mesh number is 500 mesh, and exit is equipped with bottom diameter cell screen clothes and cell strainer, and bottom diameter cell screen clothes mesh number is 50 mesh, cell filter Mesh number is 200 mesh, and buffer area is also equipped between bottom diameter cell screen clothes and cell strainer, is conducive to the stability of system circulation.

8. monkey according to claim 1-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In the plasma separator hollow fibre filter membrane diameter is 270 μm~370 μm, and film thickness is 50 μm, and aperture is 0.2 μm~0.6 μm, fibre length is 13.5 μm~26 μm, can filter blood plasma but cannot filter all cell components.

9. monkey according to claim 1-people's cell merges the preparation of female tire blood group incompatibility treatment hybrid strain, feature exists In the pathogenic antibody adsorbing therapy device includes one end of arterial blood line pipe (1) through heparin pump (2) and blood pump (3) and blood It starches separator (4) to be connected, plasma separator (4) absorber (8,9) in parallel with two through blood plasma pump (6) and circulation line (7) It is connected, is then successively connected with circulation line (10), venous line (5).