CN1134537C - Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population - Google Patents
Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population Download PDFInfo
- Publication number
- CN1134537C CN1134537C CNB991175891A CN99117589A CN1134537C CN 1134537 C CN1134537 C CN 1134537C CN B991175891 A CNB991175891 A CN B991175891A CN 99117589 A CN99117589 A CN 99117589A CN 1134537 C CN1134537 C CN 1134537C
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- stem cell
- human
- cell population
- primitive mesenchymal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Abstract
The present invention discloses a method for the separation, the in vitro culture, the preparation and the application of human primitive mesenchymal stem cell groups. An immunity method through anti-human CD45 antigens and anti-CD39 antibodies is used, and human primitive mesenchymal stem cell groups are separated by a magnetic field. An optimization culture system with low serum concentration in vitro is used for culture, and bone cells, cartilage cells, fat cells, mechanocyte, tendon stroma cells, marrow stroma cells, particularly smooth muscle cells, vascular endothelial cells and other human primitive mesenchymal stem cells groups, which have mesenchymal issues with the self-update capability, are obtained through induced multi-direction differentiation. Quick augmentation can be realized. The human primitive mesenchymal stem cell groups and human primitive mesenchymal stem cell groups with modified genes can be widely applied to human bodies clinically.
Description
The present invention relates to have multidirectional differentiation potential, can be divided into the human primitive mesenchymal stem cell population with self ability (Human Totipotent Mesenchymal Stem Cells is abbreviated as HTMSC) of multiple mesenchymal tissues such as skeletonization, cartilage, fat, unstriated muscle, tendon, endotheliocyte.Relate to can be in the human clinical widespread use, comprise bone repair, improve myatrophy, after the genetic flaw disease (hemophilia A, B, mucopolysaccharidosis etc.), radiotherapy, chemotherapy and hinder human primitive mesenchymal stem cell population behind patient's the microcirculatory reconstruction of marrow, short hematopoiesis, enhancing immunity, vaccine, antineoplastic human primitive mesenchymal stem cell population and the genetic modification again.The preparation method who particularly relates to the human primitive mesenchymal stem cell population behind a kind of human primitive mesenchymal stem cell population and the genetic modification.
The human stem cell cording has the initiating cell of the multidirectional differentiation of self, comprises that (1) from embryo or the isolating embryo of tire hepatic tissue stem organization, can be divided into the various tissues of human body; (2) from adult's marrow isolated hematopoiesis stem organization, can form lymph, grain system, red be various hematopoietic cells and brain cell; (3) from the isolating neuron stem cell of tire brain, can be divided into neuron and neuroglia; (4) can be divided into bone, cartilage, muscle, tendon etc. from adult's isolating mesenchymal stem cell of marrow (Mesenchymal Stem Cells).In recent years, peculiar biological property of stem cell and potential source biomolecule are learned using value becomes field of biology hottest point problem, it is surplus April that SCIENCE (in November, 1998) has reported that the original embryonic stem cell of in-vitro separation can be kept its undifferentiated state, and can be divided into various human tissue cells.SCIENCE (in March, 1999) has reported that once more embryonic stem cell has its limitation: (1) external utmost point is difficult to simulate microenvironment makes embryo stem cell for directional be divided into required tissue; (2) embryonic stem cell has sizable tumour generation potentiality.Therefore, increasing scientific experiment shows, mesenchymal stem cell has than thinking in the past that more muching actual biological applications was worth.
Former studies shows: the human mesenchymal stem cell cording has the raw bone myelocyte of the multidirectional differentiation and proliferation ability of self.The inside and outside can be divided into scleroblast [Haynes Worth, S.E.etal, Bone, 1992 at more suitable environment; 13:69-80], chondrocyte [Johnstone, B.etal, Ortho.Res.Soc., 1996; 21:65], adipocyte [Pittenger, M.F.etal, Mol.Biol.Cell., 1996; 7:305], smooth muscle cell, inoblast, marrow stromal cell and multiple vascular endothelial cell.But early stage mesenchymal stem cell inside and outside long term growth.
The separation of human mesenchymal stem cell adopts known mesenchymal stem cell surface marker more in the world at present, and employing STRO-1 monoclonal antibodies such as Simmon are identified as fiber colony forming cell (CFU-F), the called after stroma stem cell.STRO-1 male stroma cell can be divided into Reticulocyte, adipocyte and bone cells.This cell has CD10, CD13, CDW90, CD106 surface marker, the isolating mesenchymal stem cell of this method has been confined to only enrichment STRO-1 male human mesenchymal stem cell, the STRO-1 positive rate only accounts for mesenchymal stem cell 5-10%, and the overwhelming majority especially primitive mesenchymal stem cell may lose in sepn process.Caplan etc. have cloned SB1 to SB5 series monoclonal antibody and can discern a leaf progenitor cell and be divided into the scleroblast of different times, and have cloned SH2, SH3 and SH4 antibody with leaf progenitor cell between identification, and this cell can be divided into bone, cartilage, fat etc.This focuses on that bone takes place and applied research.
Human mesenchymal stem cell research preparation method's defective was in the past: isolating mesenchymal stem cell can not be divided into endotheliocyte, smooth muscle cell in the mesenchymal tissue through vitro culture.In other words, isolated cells may not contain than the primary stem cell, primary human mesenchymal stem cell with smooth muscle cell, endothelial cell differentiation ability, the stem cell of present method development, the human primitive mesenchymal stem cell that promptly has smooth muscle cell and endothelial cell differentiation potential, often become the positive mesenchymal stem cell of osteocyte specific transcription factor, the CBFA1 positive can be divided into scleroblast progenitor cell, chondrocyte's progenitor cell, adipocyte progenitor cell, inoblast progenitor cell, stroma cell progenitor cell.With reference to Fig. 1.
Research shows in the body: but mesenchymal stem cell self bone marrow microenvironment not only, people's marrow hemopoiesis microenvironment is provided, include and help that hematopoiesis is done, progenitor cell differentiation, propagation and express, secretion adhesion factor and Hemopoietic factor, and can be divided into various mesenchymal tissues.Shown that mesenchymal stem cell has the various biological using value.Therefore, external set up a kind of separate and the preparation system of cultivator primitive mesenchymal stem cell population most important.
One of purpose of the present invention is for isolating the human primitive mesenchymal stem cell population with endotheliocyte and smooth muscle cell differentiation capability first in the world from the medullary cell of arbitrary common human body or cord blood cell; Another object of the present invention is the culture system long term culture human primitive mesenchymal stem cell population of optimizing by outside human body, can keep the virgin state of mesenchymal stem cell to greatest extent, but and in the substratum of special component induced orientation differentiation preparation become multiple mesenchymal cell; A further object of the present invention be preparation a kind of in clinical, have comprising of widespread use can therapeutic gene after the defective disease, chemotherapy, radiotherapy and hinder the reconstruction of patient's bone marrow microenvironment, bone repair, the myatrophy of improving again, and short hematopoiesis, enhancing immunity, vaccine, the human primitive mesenchymal stem cell population of multidirectional purposes such as antitumor and the human primitive mesenchymal stem cell population behind the genetic modification.
The objective of the invention is to realize by following method:
One, the separation of human primitive mesenchymal stem cell population
1, the separation of mononuclearcell:
Gather the medullary cell or the cord blood cell of common human body, through 1600 rev/mins of medullary cells of density gradient centrifugation 20 minutes, obtain single bony nodule myelocyte, after containing 1% bovine serum albumin phosphoric acid buffer and cleaning cell 2 times, suspension cell is in phosphoric acid buffer.
2, the primary dcreening operation of human primitive mesenchymal stem cell population:
Adopt bag by the magnetic bead of anti-rat immune globulin M antibody with after the monoclonal antibody of mouse-anti people CD45 antigen and anti-CD39 combines, again with BMNC room temperature effect half an hour, to be positioned in the groove of magnetic field with the medullary cell of saturation concentration specific antibody reaction, through phosphoric acid buffer flushing and collection and the isolating medullary cell of magnetic bead.
The medullary cell that contains human primitive mesenchymal stem cell population further is marked with the fluorescein antibody response with anti-people CD45, CD39 and CD14, through flow cytometry analysis, separates and removes CD45, CD14 and CD39 male medullary cell.This process has been removed the sophisticated lymphocyte of marrow, macronucleus, monokaryon and red corpuscle parent cell etc.Collection contains the cell of the mature cell phenotype feminine gender of human primitive mesenchymal stem cell population, and this cell mass can be divided into large volume and small volume cell, and the small volume cell accounts for 2%, and the large volume cell is a human primitive mesenchymal stem cell, accounts for medullary cell 0.03-0.06%.
Characteristics: the preparation method of this separation of human primitive mesenchymal stem cell, the separation method of human mesenchymal stem cell different from the past.Use monoclonal antibody such as SH2, SH3, the STRO-1 etc. of specific recognition human mesenchymal stem cell in the past, only limited to separate SH2, SH3, STRO-1 male human mesenchymal stem cell.This mesenchymal stem cell can break up becomes scleroblast, adipocyte, stroma cell, chondrocyte etc., but does not still have the report of smooth muscle cell and endothelial cell differentiation potential.This separation method separation method different from the past is not limited to and collects SH2, SH3 or STRO-1 male stem cell.Separation and concentration of the present invention not only has mesenchymal stem cell differentiation potential in the past, and can break up the primitive mesenchymal stem cell that becomes smooth muscle cell, vascular endothelial cell.Referring to Fig. 2: the separation of human primitive mesenchymal stem cell population, cultivate propagation and multidirectional differentiation.Wherein 1. for gathering human bone marrow cell or cord blood cell, 2. obtain the mononuclearcell layer for density gradient centrifugation, 3. be the magnetic field groove, 4. go out to contain people's mesenchymal cell medullary cell for immunosorption at magnetic field separation, 5. has mesenchymal cell phenotype cell for small volume, 6. be the large volume human primitive mesenchymal stem cell population, phenotype is that CD13, CD14, CDW90, IB10, activation are from the cell adhesion molecule positive; CD50, CD39, DR, CD45, CD68 feminine gender 7. are flow cytometer: be used for analysis, the fluorescently-labeled mesenchymal cell of separating immune.
Two, the vitro culture of human primitive mesenchymal stem cell population and multidirectional differentiation, propagation
1. the vitro culture of human primitive mesenchymal stem cell population
The human primitive mesenchymal stem cell that obtains is positioned over the culture plate cultivation of wrapping quilt through Fibronectin (FN).Comparative studies three kinds of different culture condition, comprising: 1. 2. serum-free contains the culture system that 3. low calf serum contains 12% calf serum, 12% horse serum.The result shows: 1. serum-free only contains thrombocyte derivation somatomedin, endothelial cell growth factor (ECGF), dexamethasone, selenium, Transferrins,iron complexes, Regular Insulin, linolic acid, bovine albumin, xitix, antibiotic DMEM and MCDB substratum and 2. contain 12% calf serum, horse serum, the culture condition of hydrocortisone and β thioglycol is unfavorable for that the human primitive mesenchymal stem cell population body does not have the cell growth of differentiation.Containing low calf serum, dexamethasone, BB thrombocyte derivation somatomedin and endothelial cell growth factor (ECGF), selenium, Transferrins,iron complexes, Regular Insulin, linolic acid, albumin, xitix, 10 milliliters/rise antimycoin ,/10,000 unit penicillin ,/the DMEM culture medium culturing of 10,000 microgram Streptomycin sulphates, 25 micrograms, two property key element B, F-12 nutritional blend, the external long term growth of human primitive mesenchymal stem cell population and keep undifferentiated state and be the human primitive mesenchymal stem cell phenotype.Cell is the spindle bodily form, changes the broth out suspension cell, and carries out passage with 0,25% trypsin digestion cell that contains 1 mmole EDTA in per 4 days.Human primitive mesenchymal stem cell population cellular immunization phenotype is CD13, CD44, CD49b, CDW90,1B10, the activated leukocyte adhesion molecule positive, CD50, CD39, DR feminine gender.
Former studies shows that SH2, SH3 or STRO-1 are positive, and mesenchymal stem cell accounts for ten thousand single bony nodule myelocytes of 1/1-10, and the present invention adopts the limiting dilution method to determine the about 1/1-5 1,000,000 single bony nodule myelocytes of human primitive mesenchymal stem cell population.Culture condition adopted the culture condition of 10% serum to cultivate mesenchymal stem cell in the past.The present invention adopts than the culture condition of low-serum-concentration near the human physiology condition, to keep the growth in vitro that optimum cell does not break up virgin state.
2, the multidirectional differentiation of human primitive mesenchymal stem cell population.
Through 4 be commissioned to train support after, human primitive mesenchymal stem cell population can multidirectional differentiation become scleroblast, chondrocyte, adipocyte, stroma cell, myocyte and endotheliocyte etc., and wherein smooth muscle cell and endothelial cell differentiation potential are that mesenchymal stem cell did not appear in the newspapers in the past.Referring to Fig. 1.
1. differentiation becomes scleroblast: through containing dexamethasone, xitix, β phospho-glycerol and β transforming growth factor superfamily albumen (as bone morphogenetic protein 2,3), somatomedin such as fibroblast growth factor, the culture system of prostaglandin(PG) etc. was cultivated after 7 days, alkaline phosphatase peaking in the cell.After 14 days, Von Kossa dyeing and fluorescence calcium bone mineralising are measured human mesenchymal stem cell differentiation becoming scleroblast.Leaf material between scleroblast secretion and formation bone like cell.
2. differentiation becomes the chondrocyte: cultivated 2-3 days through containing β transforming growth factor or statin A, cartilage stimulating activity factor bone morphogenetic protein 4 etc., histology is identified: the Toluidine blue staining positive is the chondrocyte.Immunohistochemical analysis, the chondrocyte indicates the CSPG-M positive.
3. differentiation becomes endotheliocyte: after containing human vascular endothelial growth factor and cultivating for 2 weeks, to be that CD36, CD34 and the Von Willerband factor are positive be endotheliocyte to cell phenotype.
4. differentiation becomes the myocyte: after containing 5-azacytidine and inducing 1 day, 2 week back cellular immunization group mensuration, the troponin positive is the myocyte.In the atomization, multiple transcription factor such as MyoD, Myf5, Myf6 and Myogenin express the positive respectively in early days.
5. differentiation becomes stroma cell: at inducing culture cultivator primitive mesenchymal stem cell populations such as the specific cell factor such as interleukin-11 α or interleukin-22s, the cultivation of going down to posterity of 30% cell density, induced differentiation through 3 days, collect and filter supernatant, measure the new excretory cytokine that has been divided into stroma cell, comprise that interleukin-13,6, G-CSF, GM-CSF, leukaemia inhibitory factor, stem cell factor, β 2 change for somatomedin.Through the quite high-caliber above-mentioned cytokine of inductive stroma cell secretion, and undifferentiated cell only the interleukin 6 expression level is higher.
6. differentiation becomes adipocyte: containing dexamethasone, 1 methyl, 3 isobutyl xanthine, Regular Insulin and indomethacin, calf serum substratum inducing culture mesenchymal stem cell group 2-3 days, after 1 day, begin form lipid cavity after the 2nd day through insulin-containing and former calf serum and nutritive ingredient substratum then.
Characteristics: rapid, the homogeneous differentiation of stem cells evolution of external evoked differentiation of human primitive mesenchymal stem cell population system, this cell engineering has widespread use and is worth.The human primitive mesenchymal stem cell population of a, purifying does not contain the cell biological factor that is unfavorable for multidirectional differentiation; B, externally can control the cytodifferentiation phase, especially be the bone different development stage; C, externally simulate required noble cells hypotype, form fast or slow muscle cell as muscle; D, vitro differentiation homogeneous, rapid, human primitive mesenchymal stem cell population are exposed to suitable dosage simultaneously and induce biologically active factors with directed differentiation, and often induce differentiation gradually for the part microenvironment in the body, last length, have polymorphic minute voltinism.
Human primitive mesenchymal stem cell population mesenchymal stem cell more in the past has more differentiation potential, comprises smooth muscle cell and endotheliocyte, and the rapid vitro differentiation advantage of homogeneous realizes widespread use for it and established solid foundation in addition.
3, the growth in vitro of human primitive mesenchymal stem cell population
Human primitive mesenchymal stem cell population propagation can be bred 1,000 times the 1st week rapidly, can breed 30,000 times the 4th week, can breed about 1 thousands of times the 8th week.The human primitive mesenchymal stem cell population observation in vitro surplus undifferentiated state that still keeps in February is exponential growth.Referring to Fig. 2.Wherein 8. are Tissue Culture Dish: be used for the FN bag by the cultivator primitive mesenchymal stem cell population, changed liquid in per 4 days, to remove suspension cell, 9. be spindle body for the human primitive mesenchymal stem cell population microscopically, has the mesenchymal stem cell phenotype, 10. can increase 1,000 times for first week of human primitive mesenchymal stem cell population, the 8th week can reach 1 thousands of times, 11. for being induced to differentiate into scleroblast, 12. for being induced to differentiate into the chondrocyte, 13. for being induced to differentiate into endotheliocyte, 14. for being induced to differentiate into the myocyte, 15. for being induced to differentiate into other histocytes.
Three, the human primitive mesenchymal stem cell population behind the preparation genetic modification
Adopt retroviral vector such as MSCV-IRES-EGFP.Characteristics are: 1, have than strong promoter and open the beginning foreign gene, especially efficiently express at hemopoietic stem cell; 2, overcome functional gene Silencing shortcoming due to the first-generation retroviral vector; 3, this carrier has kept the advantage of secretion infectious titer supernatant.
Adopt internal ribosome to open beginning initial expressive function gene of sequence (IRES) and screening-gene, can make single promotor translate the upstream and downstream gene of IRES guiding simultaneously, express to obtain the par double protein, overcome in the past the carrier endogenesis promoter from phase arrestin expressional function.
Adopt best screening-gene green fluorescent protein (GFP), through the enhancing green fluorescent protein (EGFP) of 64,65 site mutations.Characteristics are: need not reaction substrate, fluorescence intensity increases to 100 times, can obtain to contain the fluorescencepositive cell of the modification of gene expression, can fast separate through flow cytometer in 24 hours, applied research.
Modifying factor can be multidirectional induction gene (bone morphogenetic protein gene, β transforming growth factor etc.), genetic flaw disease missing gene (factor V lII, IX hemophilia gene, mucopolysaccharidosis gene I S etc.) and short hematopoiesis, the cytokine of enhancing immunity, Antioncogene.As shown in Figure 3, with the modifying factor clone, after cutting, enzyme is connected in upstream BglII or XhoI, downstream EcoRI restriction enzyme site.Referring to Fig. 3: retroviral vector design, cotransfection and enrichment infectious titer supernatant transduction human primitive mesenchymal stem cell population.Wherein X. represents arbitrary gene with the transduction human primitive mesenchymal stem cell population, and B. is Bgill or Xho 1 restriction enzyme site, and E. is EcoR 1 restriction enzyme site, 1. be MSCV reverse transcription disease virus promoter, 2. treating row for the IRES internal ribosome opens the beginning, for EGFP strengthens green fluorescent protein, 4. is the CMV promotor 3..
Four. preparation contains the infectious titer supernatant and the gene transfection human primitive mesenchymal stem cell population of modifying factor
1. adopt the quick transfection system of PCL-Ampho plasmid, with retroviral vector cotransfection 293Kj cell, PCL and virus vector 20 microgram plasmid DNA are added 725 microlitres (0.25M) calcium chloride and (PH7.0) 2 times of HBS damping fluids of 725 microlitres mixing, again with 200 ten thousand 293Kj co-culture of cells (37c) 6 hours, take out transfection liquid and add 5 milliliters of 15% glycerine damping fluids, shock cell 1 minute, add 5 milliliters of nutrient solution culturing cells, second and third day collected the viral supernatant liquor that contains modifying factor.
2. adopt VSV-G plasmid enrichment virus system, behind virus vector and PCL plasmid co-transfection, the viral supernatant of collecting was through the super centrifugal 50000g of Beckman L3-50 (4c) 90 minutes, precipitate 12 hours with 4 ℃ of lytic viruses of 0.1%Hanks liquid then, can concentrate 200-2000 doubly through this system's virus, viral supernatant titre can reach 2 * 10
9(colony forming unit/milliliter).
3. gene transfection
Adopt the glue primordial covering to change pore membrane culture plate, streaming virus transfection method.The hole device is changeed in 0.4 μ m hole of glue primordial covering be positioned over Tissue Culture Plate, the virus supernatant continues percolation and changes film, with enrichment virus supernatant, with transfected co-culture of cells 24 hours 2 times, intermediate phase was every 12 hours, and cell cultures and gene transfection whole process are carried out at low blood serum medium.This virus transfection can obtain human mesenchymal stem cell gene transfection positive rate up to 20-50%.Referring to Fig. 3.Wherein 5. is that VSV-G is in order to enrichment virus supernatant, 6. being CMV LN enhanser promotor, 7. is the gag gene, 8. is the pol gene, 9. be the Ampho gene, 10. be the 293kj cell, 11. is the L3-50 ultracentrifuge, and 12. are the virus of enrichment, 13. for changeing the porocyte culture plate, 14. be that human primitive mesenchymal stem cell population is cultivated altogether with virus, transfectional cell, 15. for the human primitive mesenchymal stem cell population of genetic modification feeds back to the patient again, to satisfy the various clinical purposes.
Five. preparation is widely used in clinical human primitive mesenchymal stem cell population
1. preparation is applied to the human primitive mesenchymal stem cell population behind the genetic modification of bone healing
In the bone growth and development through three main processes: cell chemotaxis, mitotic division and differentiation become scleroblast.Wait after the wound and can bring out the acquired character bone and build again, but bone morphogenetic protein induced osteogenesis progenitor cell has been oriented osteoprogenitor cells needing no foreign signal and can have formed preosteoblast, scleroblast and osteocyte.
Preparation is applied to the human primitive mesenchymal stem cell population behind the genetic modification of bone healing, the enrichment human primitive mesenchymal stem cell, select the induced osteogenesis cell to generate culture medium culturing, and will have the induced osteogenesis cell gene (bone morphogenetic protein 2 or 4 or Regular Insulin generate the factor etc.) clone in the upstream of IRES MSCV-IRES-EGFF carrier and become the MSCV-X-IRES-EGFP carrier, cotransfection 293Kj cell, collect viral supernatant transduction human primitive mesenchymal stem cell population, flow cytometry analysis is collected the mesenchymal cell (being divided into scleroblast the most at last) of genetic modification.With 5 * 10
6Cell/every milliliter and weighting material be with 3 to 1 volume mixings, injects that the pathologic bone is not united, the reparation of bone delayed healing, comminuted fracture or total joint, especially causes patients' such as bone lacks bone lacks position because of autologous bone transplanting maybe can provide the bone deficiency.Weighting material is a homogeneous, the material (as hydroxylapatite and tricalcium phosphate etc.) of growth in retention mesenchymal cell group, the permission blood vessel.
2. preparation is applied to improve the human primitive mesenchymal stem cell population that the former protein gene of myotrophy of myotrophy degeneration is modified
Muscular dystrophy system carrying out property near-end myatrophy can involve heart etc.Being characterized as the blood creatine kinase increases, the parapeptone degraded, and molecular level is that unstriated muscle lacks the former albumen of myotrophy, as involves stomach and intestine and can cause hydrochloric acid in gastric juice emptying delay, acute gastric dilatation, false intestinal obstruction.
Experimentation on animals in the past shows: troponin genetic expression can be corrected the muscular dystrophy function for 50 times.To contain the troponin gene clone in the IRES upstream, tyr22DHFR gene substitution EGFP gene will suddenly change, be built into MSCV-troponin-IRES-tyr22DHFR genetic modification carrier, these carrier characteristics are: the tyr22DHFR gene is first hydrogen folic acid (MTX) drug resistant gene (Zhao, RCH., etal, Blood, 1997; 12:4687), can pressurize to induce causes that this troponin-IRES-tyr22DHFR gene examines the shellfish number and increase, and corrects muscular dystrophy to obtain gene high expression.
Place the preferable myocyte that induces to generate culture medium culturing human primitive mesenchymal stem cell population, employing contains MSCV-troponin-IRES-tyr22DHFR gene viruses supernatant transduction mesenchymal cell, be that virus is examined shellfish and people's mesenchymal cell 20 to 1, cultivate 24 hours secondaries altogether containing the low blood serum medium of 8 microgram protamine, intermediate phase was every 12 hours.Contain the foetal calf serum substratum through first hydrogen folic acid (0.25 micromole) and screened for 2 weeks, the human primitive mesenchymal cell mass that contains troponin modification myocyte and the fusion of pathologic myofiber that makes that can finally correct this carrier of expression of muscular dystrophy again is expelled to muscular dystrophy patient's affected part.
3. preparation is applied to the human primitive mesenchymal stem cell population behind the genetic modification of genetic flaw disease
1. hemophilia A, B are the hemorrhagic diseases due to blood coagulation factor VIII, IX lack.Heavy hemophilia thrombin is lower than normal level 1%.Blood plasma or recombinant factor replacement therapy are main treatment means at present.The deficiency of this conventional treatment is: the transformation period is short in medicine costliness and the thrombin body.2. mucopolysaccharidosis is that fatal lysosome is stored up imbalance, owing to lack serious bone due to the IDS, nervous symptoms.Unique treatment is symptomatic treatment and bone marrow transplantation, but curative effect does not show.3. Cysticfibrosis: be that cystic fibrosis is changeed due to the film modulin defective.4. several genes defective disease all needs to set up somatic cell gene expression-secretion steady in a long-term system.Human primitive mesenchymal stem cell population possesses external a large amount of amplification, and has that the inside and outside breaks up for a long time, multiplication characteristic, but associating retrovirus stable integration somatic cell gene group chromosome DNA overcomes retrovirus and is difficult for transduction non-proliferative target cell weakness.
This preparation method adopts the MSCV retrovirus to have and stronger opens primordium because of effect, can 10 times efficiently expresses in hemopoietic stem cell, and overcomes gene silencing deficiency.Lower level is expressed and can be corrected genetic flaw, even also non-evident effect of integrator gene high level expression.With the normal gene-IRES-EGFP transduction human primitive mesenchymal stem cell population of MSCV patient's defective, the screening positive gene carries cell, and freeze-stored cell quantitatively feeds back and gives the patient,
4. preparation is applied to the human primitive mesenchymal stem cell population after bone marrow microenvironment is rebuild the genetic modification that reaches short hematopoiesis
1. the patient works a large amount of marrow stromal cell damaged of supporting marrow hemopoietic stem cells after a large amount of radiotherapies, chemotherapy, can not secrete essential Hemopoietic factor and the important adhesion factor of expression to keep the normal bone marrow microenvironment.The reconstruction of bone marrow microenvironment is to help hemopoietic stem cell marrow to regrow, break up vital factor, also is the key that the patient spends early stage critical days after the bone marrow transplantation.
Before the patient is carried out a large amount of radiotherapies, chemotherapy, gather patient's self marrow, place vitro culture, proliferation of human primitive mesenchymal stem cell population, frozen, treat to feed back to the patient again after the bone marrow transplantation.
2. hinder patient's hematopoieticmicroenviron-ment again and be subjected in various degree destruction.Gather human primitive mesenchymal stem cell population, the virus vector of clone's construction expression human interleukin-13 factor, transduction MSCV-interleukin-13-IRES-EGFP gene feeds back to hindering the patient in human primitive mesenchymal stem cell population more again.
3. the anaemia that causes of anaemia, especially chronic disease (as diseases associated with inflammation, rheumatic arthritis, severe infections, tumour etc.).Employing contains the retrovirus transduction human primitive mesenchymal stem cell population of erythropoietin, feeds back the patient who causes anaemia to chronic disease again.
5. preparation is applied to the human primitive mesenchymal stem cell population behind the genetic modification of purposes such as enhancing immunity, tumor vaccine
The gene therapy of tumor vaccine relates to the immune regulation and control of cell and molecular level, and the adjusting of histocyte propagation, differentiation, living or death.But most tumors is through factor inductor internal specific tumour antigens such as the transducer cell factor such as GM-CSF, interleukin-22,4,6, Interferon, rabbit, to strengthen identification and specific killing in the body: as melanocytoma specific tumour antigen Magel, Martl, gpioo, the abduction delivering of tyrosine oxidase.Existing gamma-interferon has been widely used in trichoblast leukemia, chronic myelocytic leukemia (slow grain), Kaposi sarcoma, malignant melanoma etc.
1. chronic myelocytic leukemia is the hemopoietic stem cell malignant tumour, a large amount of in, metamyelocyte is at the peripheral blood malignant proliferation.Adopt human primitive mesenchymal stem cell population to have the early stage medullary cell of normal hematopoiesis, immunological competence, after cytokine is modified, the MSCV-alpha-interferon IRES-EGFP virogene transduction human primitive mesenchymal stem cell population that will contain alpha-interferon genes feeds back and comprises slow granulosis person for immunity function tumour low or that immune cognitive disorders caused.
2. adopt same scheme, the human primitive mesenchymal stem cell population with behind the employing gamma-interferon genetic modification feeds back patients such as giving chronic myelocytic leukemia, trichoblast leukemia again.
3. adopt same scheme, the human primitive mesenchymal stem cell population with behind employing transduction interleukin-22, the gamma-interferon genetic modification feeds back the patient who gives the whole body metastatic tumo(u)r again.
Six, human primitive mesenchymal stem cell population preparation method's example is done
1, obtains patient's marrow primitive mesenchymal stem cell population
1. separate single bony nodule myelocyte
Gather patient's self medullary cell, through 1600 rev/mins of medullary cells of density gradient centrifugation 20 minutes, obtain single bony nodule myelocyte, after containing 1% bovine serum albumin phosphoric acid buffer and cleaning cell 2 times, suspension cell is in phosphoric acid buffer.
2. enrichment patient primitive mesenchymal stem cell population
Adopt bag by the magnetic bead of anti-rat immune globulin M antibody with after the monoclonal antibody of mouse-anti people CD45 antigen and anti-CD39 combines, again with BMNC room temperature effect half an hour, to be positioned in the groove of magnetic field with the medullary cell of saturation concentration specific antibody reaction, through phosphoric acid buffer flushing and collection and the isolating medullary cell of magnetic bead.The medullary cell that will contain patient's primitive mesenchymal stem cell population further is marked with the fluorescein antibody response with anti-people CD45, GlyA and CD14, through flow cytometry analysis, separates and removes CD45, CD14 and GlyA male medullary cell.This process has been removed the sophisticated lymphocyte of marrow, macronucleus, monokaryon and red corpuscle parent cell etc.Collect patient's primitive mesenchymal stem cell population.
2, the cultivation of external patient's primitive mesenchymal stem cell population
The patient's primitive mesenchymal stem cell population that obtains is positioned over the culture plate cultivation of wrapping quilt through FN.Containing low calf serum, dexamethasone, BB thrombocyte derivation somatomedin and endothelial cell growth factor (ECGF), selenium, Transferrins,iron complexes, Regular Insulin, linolic acid, albumin, xitix, 10 milliliters/rise antimycoin ,/10,000 unit penicillin ,/the DMEM culture medium culturing of 10,000 microgram Streptomycin sulphates, 25 micrograms, two property key element B, F-12 nutritional blend, the external long term growth of patient's primitive mesenchymal stem cell population and keep undifferentiated state.
3, the clone makes up MSCV-IFN-α-IRES-EGFP retroviral vector
After patient's alpha-IFN gene downstream end enzyme cut,, mended flatly in 20 minutes, after DNA is purified, cut the alpha-IFN gene upstream, obtain 677bp (XhoI--flush end) alpha-IFN gene with the XhoI enzyme with 4 ℃ of Klenow enzyme reactions.Equally, the MSCV carrier is cut back Klenow enzyme benefit with the EcoRI enzyme and is put down, and cuts through the XhoI enzyme and produces the connection site that is complementary.Alpha-IFN gene is cloned into the MSCV retroviral vector of XhoI--flush end, and dna sequence analysis confirms to obtain MSCV-IFN-α-IRES-EGFP carrier.Referring to Fig. 4.
4, obtain the viral supernatant that high titre contains interferon-alpha
External a large amount of amplification MSCV-IFN-α-IRES-EGFP, MSCV and PCL plasmid DNA, with the above-mentioned DNA mixing of 20 micrograms in the calcium chloride of 0.25 mole of 725 microlitre, again with 725 microlitre HBS damping fluids (8.2 gram sodium-chlor, 5.9 gram HEPES, 0.21 the gram Sodium phosphate dibasic adds water to 500 milliliters) the even even and fine DNA throw out that forms shakes.
With 2 * 10
6Cell/per 10 milliliters of DMEM culture medium culturing 293Kj cells are in 10 cm diameter culture dish of gelatin bag quilt, treat that cell density is 40% next day, above-mentioned DNA throw out is added 37 ℃ of 293Kj co-culture of cells, 6 hours, remove cells and supernatant, add 5 milliliters and contain 15% Phosphoric acid glycerol esters damping fluid room temperature function cells 1 minute, after washing the 293Kj cell with phosphoric acid buffer, add 5 milliliters of DMEM substratum and be positioned over 37 ℃ 5% carbonic acid gas incubator culturing cell, collect viral supernatant after the 1st, 2 day.
5, the interferon-alpha slow granulocyte series K562 that transduces suppresses the growth of its cell
The K562 cell that to be transduceed is positioned over changes the well culture plate epicoele, adds then to contain high titre interferon-alpha virus supernatant or negative control MSCV virus supernatant, and with K562 cell co-cultivation 24 hours 2 times, intermediate phase was cultivated with cell culture medium every 12 hours.After the 2nd day, the K562 cell that adopts flow cytometer to separate, express simultaneously green fluorescent protein and interferon-alpha reaches the only MSCV control group K562 cell of green fluorescent protein expression, observes the growth effect of interferon-alpha to the K562 cell.
1. PCR detects the K562 cell alpha-IFN gene of interferon-alpha transduction
Design interferon-alpha upstream primer, 5 '-CAG TTC CAG AAG GCT CAA GC-3 ' and downstream primer 5 '-ACC TCC TGC ATC ATA CAG GC-3 ' and endogenous crt gene β-Actin upstream and downstream primer, the K562 cell DNA of extraction and purifying interferon-alpha or MSCV transduction, external at 95 ℃, 1 minute; 58 ℃, 1 minute; 72 ℃, 1 minute; 30 circulations were extended 7 minutes for back 72 ℃, with the amplification alpha-IFN gene.The interferon-alpha transduction K562 cell 173bp length alpha interferon gene that can increase, and the MSCV control group is negative.Confirm that alpha-IFN gene has been carried in the K562 cell DNA.
2. interferon-alpha suppresses the growth of K562 cell
Observe the growth of interferon-alpha transduction K562 cell, control group adopts MSCV transduction K562 cell, with slow granulocyte series K562 cell with 1 * 10
512 porocyte culture plates are placed in/every hole, continue observation of cell propagation.Measure 1 to 4 day cell proliferating number, get the blue dyeing of 50 microlitre cell suspensions and placenta active cells counting at every turn.The result shows: the K562 cell cell proliferation in the 1st day of control group and interferon-alpha transduction quite is 12 * 10
4/ every milliliter, beginning to be suppressed from the growth of the 2nd day interferon-alpha transduction K562 cell, cell was significantly suppressed in the 4th day, and the cellular control unit number is 90 * 10
4/ every milliliter, and interferon-alpha transduction K562 cell only 30 * 10
4/ every milliliter.
6, interferon-alpha transduction marrow CD34 positive cell unrestraint colony formation effect
Employing with alpha-IFN gene transduction of CD 34 positive cells, separates interferon-alpha positive marrow CD34 cell through flow cytometer with quadrat method.Observe the influence that alpha-IFN gene forms marrow CD34 positive cell colony.Control group is the CD34 positive cell of MSCV transduction, measures the growth of hematopoietic cell CFU-GM colony and adopts CD34 male 5000 cells to be positioned over the semi-solid agar culture system that contains 10 nanogram(ng)s/milligram interleukin 6, interleukin-13, G-CSF and 3 units/every milliliter of erythropoietin.Through 37 ℃ contain 5% carbonic acid gas incubator and cultivated for 2 weeks after, under inverted microscope, observe GM colony forming unit and BFU-E (red is colony forming unit) and counting.The result shows: control group is suitable with interferon-alpha group colony, is about 250 units.The prompting alpha-IFN gene does not have obvious marrow CD34 positive cell colony and forms restraining effect.
7, interferon-alpha transduction human primitive mesenchymal stem cell population
Interferon-alpha virus supernatant in order to the transduction human primitive mesenchymal stem cell population, is obtained 38% efficient transduction human primitive mesenchymal stem cell.Through the positive patient's primitive mesenchymal stem cell population of flow cytometer enriching of alpha Interferon, rabbit, frozen, feed back again in batches and give slow granulosis person.
Advantage of the present invention is:
1. separate human primitive mesenchymal stem cell population simple and direct, feasible, repeat.
2. human primitive mesenchymal stem cell population can external a large amount of amplifications, and the 8th week is 1 thousands of times nearly, and still Keep former mesenchymal stem cell differentiation, proliferation potential and phenotype.
But the human primitive mesenchymal stem cell population Multidirectional Differentiation become Gegenbaur's cell, cartilage cell, myocyte, Adipocyte, fibroblast, marrow stromal cell, and reported first of the present invention can be divided into Multiple vascular endothelial cell and smooth muscle cell etc.
4, human primitive mesenchymal stem cell population is taken from bone marrow cell or the cord blood cell of common human body, Overcome the source difficulty of embryonic stem cell, and external can't inducing is divided into specific tissue etc. no Foot, the mesenchymal tissue cell that can provide again multiple candidate stem cell to provide.
5. human primitive mesenchymal stem cell population is effective, feasible gene therapy system in conjunction with retrovirus. Can modifier of expressing through viral integrase human body cell chromosomal DNA steady in a long-term, reach gene therapy Purpose. Human primitive mesenchymal stem cell population is bred the dyeing of permission retrovirus stable integration human genome in a large number Body DNA; After the transfection, this cell still keeps the original differentiation of mesenchymal stem cell, proliferation potential, and the inside and outside can The gene of modifying of expressing steady in a long-term. Also overcome adenovirus, adeno-associated virus (AAV) (AAV) seldom Transducible gene human gene group DNA's deficiency, and adverse immune response in the body due to this viral product. Overcome Lenti virus and be not good at the deficiencies such as safety.
6, MSCV retroviral vector tool efficiently expresses modifier in stem cell, no silencing; Adopting IRES not have due to the endogenesis promoter gene expression disturbs; Select the enhancing green fluorescent protein fast to separate The positive cell that contains modifier.
7, adopt the viral system of quick transfection enrichment, can obtain viral supernatant up to 2 * 10 in three days9。
8. adopting to be coated with turns to hole incubator streaming virus transfection, can obtain gene transfection human mesenchymal stem cell rate Up to 20-50%.
9. human primitive mesenchymal stem cell population can be induced Multidirectional Differentiation, is applied to bone repair, smooth muscle gene Treatment, hematopoieticmicroenviron-ment reconstruction, the treatment of gene defect disease gene, anti-tumor vaccine, enhancing hematopoiesis, The multiple uses such as immunity.
Claims (8)
1, the preparation method of a kind of human primitive mesenchymal stem cell population (HTMSC), feature of the present invention is: the method with immunity adopts magnetic field to remove ripe medullary cell, in conjunction with the big human primitive mesenchymal stem cell of flow cytometer separated volume, cultivate through external containing than the optimization culture system of low-serum-concentration, induce multidirectional differentiation and rapidly amplification, make the human primitive mesenchymal stem cell population that is widely used in the human clinical and the human primitive mesenchymal stem cell population behind the genetic modification.
2, according to the human primitive mesenchymal stem cell population of the described preparation of claim 1, it is characterized in that: having the self ability, is to be divided into scleroblast, chondrocyte, adipocyte, inoblast, tendon, marrow stromal cell, the particularly human primitive mesenchymal stem cell population of multiple mesenchymal tissue such as smooth muscle cell, multiple vascular endothelial cell.
3, preparation method according to claim 1, it is characterized in that: extract medullary cell or cord blood cell from common human body, after centrifugal, adopt bag to be made it to combine with the monoclonal antibody of mouse-anti people CD45 antigen, anti-CD39 antibody by the magnetic bead of anti-rat immune globulin M antibody, in the groove of magnetic field, collect and the isolating medullary cell of magnetic bead, through flow cytometry analysis, remove the marrow mature cell, collect the human primitive mesenchymal stem cell of large volume.
4, preparation method according to claim 1, it is characterized in that: with the human primitive mesenchymal stem cell of collecting, place and contain lower concentration calf serum and dexamethasone, BB thrombocyte derivation somatomedin and endothelial cell growth factor (ECGF), selenium, Transferrins,iron complexes, Regular Insulin, linolic acid, albumin, xitix, antimycoin, penicillin, Streptomycin sulphate, two property key element B, cultivate in the optimization culture system than low-serum-concentration of F-12 nutritional blend, can make it outside human body, to increase in a large number and be exponential growth, form human primitive mesenchymal stem cell population, but still keep the undifferentiated virgin state of its human primitive mesenchymal stem cell.
5, preparation method according to claim 1 is characterized in that: human primitive mesenchymal stem cell population can be rapidly in the substratum of special component, the multiple mesenchymal cell of homogeneous induced orientation differentiation becoming:
1. cultivating differentiation in the culture system that contains compositions such as dexamethasone, xitix, β phospho-glycerol and β transforming growth factor superfamily albumen, somatomedin becomes scleroblast,
2. cultivating differentiation in the culture system that contains compositions such as β transforming growth factor or statin A, cartilage stimulating activity factor bone morphogenetic protein 4 becomes the chondrocyte,
3. cultivating differentiation in containing inducing culture such as the specific cell factor becomes stroma cell,
4. inducing culture differentiation becomes adipocyte in containing dexamethasone, 1 methyl, 3 isobutyl xanthine, Regular Insulin and indomethacin substratum,
5. cultivating differentiation in containing the human vascular endothelial growth factor substratum becomes endotheliocyte,
6. in containing the 5-azacytidine substratum, induce differentiation to become the myocyte.
6, preparation method according to claim 1, it is characterized in that: adopt retroviral vector, integrator gene efficiently expresses human primitive mesenchymal stem cell population, and adopts internal ribosome to open beginning sequence initial expression resistance screening-gene and adopt human primitive mesenchymal stem cell population after best screening-gene fluorescin is made genetic modification.
7, preparation method according to claim 1, it is characterized in that: adopt quick transfection system of PCL-Ampho plasmid and retroviral vector cotransfection 293Kj cell, adopt VSV-G plasmid enrichment virus system, with virus vector and PCL plasmid co-transfection and employing bag quilt commentaries on classics hole incubator streaming virus transfection method, with enrichment virus supernatant and transfected co-culture of cells, make the infectious titer supernatant that contains modifying factor and the human primitive mesenchymal stem cell population of gene transfection.
8, preparation method according to claim 1 is characterized in that: the human primitive mesenchymal stem cell population that is widely used in the human clinical of preparation and the human primitive mesenchymal stem cell population behind the genetic modification:
1. be the human primitive mesenchymal stem cell population that to modify behind the genetic modification of bone healing.The gene clone that will have the induced osteogenesis cell becomes the MSCV-X-IRES-EGFP carrier in the upstream of IRES MSCV-IRES-EGFP carrier, cotransfection 293Kj cell, through flow cytometry analysis, collect genetic modification human primitive mesenchymal stem cell population and with weighting material volume mixing in proportion, inject bone lacks patient's bone lacks part
2. be to modify the human primitive mesenchymal stem cell population that contains the former protein gene of myotrophy that improves the myotrophy degeneration.To contain MSCV-troponin-IRES-tyr22DHFR gene viruses supernatant transduction human primitive mesenchymal stem cell population cultivates in the low blood serum medium of protamine altogether, after first hydrogen folic acid contains the screening of foetal calf serum substratum, the human primitive mesenchymal stem cell population injection muscular dystrophy patient's of this carrier affected part will be expressed
3. be can therapeutic gene human primitive mesenchymal stem cell population behind the genetic modification of defective disease.Adopt the people's normal gene-IRES-EGFP transduction human primitive mesenchymal stem cell population of MSCV patient's defective; the screening positive gene carries cell; frozen; quantitatively feeding back and giving hemophilia A, B is several genes defective disease patients such as hemorrhagic diseases due to blood coagulation factor VIII, IX lack, mucopolysaccharidosis, Cysticfibrosis
4. be the human primitive mesenchymal stem cell population that can rebuild after the bone microenvironment reaches the genetic modification of urging hematopoiesis,
(1) patient's marrow before collection radiotherapy, the chemotherapy, the vitro culture human primitive mesenchymal stem cell population, frozen, treat to feed back again to the patient after patient's radiotherapy, chemotherapy, the bone marrow transplantation,
(2) virus vector of clone's construction expression human interleukin-13 factor, transduction MSCV-interleukin-13-IRES-EGFP gene are in human primitive mesenchymal stem cell population, and feedback is given and hindered the patient more again,
(3) will contain the retrovirus transduction human primitive mesenchymal stem cell population of erythropoietin, feed back the patient who causes anaemia to chronic disease again,
5. being can enhancing immunity, as the human primitive mesenchymal stem cell population of tumor vaccine,
MSCV-alpha-interferon-IRES-EGFP virogene transduction human the primitive mesenchymal stem cell population that will contain alpha-interferon genes feeds back and gives the chronic myelocytic leukemia patient; Use above-mentioned the same manner, human primitive mesenchymal stem cell population behind the employing gamma-interferon genetic modification feeds back patients such as giving chronic myelocytic leukemia, trichoblast leukemia again; Use above-mentioned the same manner, human primitive mesenchymal stem cell population behind employing transduction interleukin-22, the gamma-interferon genetic modification feeds back the patient who gives the whole body metastatic tumo(u)r again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991175891A CN1134537C (en) | 1999-09-08 | 1999-09-08 | Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991175891A CN1134537C (en) | 1999-09-08 | 1999-09-08 | Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1287166A CN1287166A (en) | 2001-03-14 |
| CN1134537C true CN1134537C (en) | 2004-01-14 |
Family
ID=5280158
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991175891A Expired - Lifetime CN1134537C (en) | 1999-09-08 | 1999-09-08 | Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1134537C (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2849201A1 (en) * | 2001-12-07 | 2003-07-03 | Macropore Biosurgery, Inc. | Adipose-derived cell processing unit |
| US20050095228A1 (en) | 2001-12-07 | 2005-05-05 | Fraser John K. | Methods of using regenerative cells in the treatment of peripheral vascular disease and related disorders |
| US7585670B2 (en) | 2001-12-07 | 2009-09-08 | Cytori Therapeutics, Inc. | Automated methods for isolating and using clinically safe adipose derived regenerative cells |
| US7651684B2 (en) | 2001-12-07 | 2010-01-26 | Cytori Therapeutics, Inc. | Methods of using adipose tissue-derived cells in augmenting autologous fat transfer |
| US8105580B2 (en) | 2001-12-07 | 2012-01-31 | Cytori Therapeutics, Inc. | Methods of using adipose derived stem cells to promote wound healing |
| US7771716B2 (en) | 2001-12-07 | 2010-08-10 | Cytori Therapeutics, Inc. | Methods of using regenerative cells in the treatment of musculoskeletal disorders |
| CN1312277C (en) * | 2004-04-29 | 2007-04-25 | 复旦大学附属中山医院 | Marrow stroma cell directional differentiation induction method |
| CN100453640C (en) * | 2006-04-29 | 2009-01-21 | 中国医学科学院血液学研究所 | Method of separating multipotent adult progenitor cells from umbilical cord blood |
| CA2682317C (en) * | 2007-04-03 | 2017-01-17 | The Cleveland Clinic Foundation | Enrichment of tissue-derived adult stem cells based on retained extracellular matrix material |
| CN109069543B (en) * | 2017-10-16 | 2020-02-18 | 中国科学院广州生物医药与健康研究院 | Method for remodeling bone marrow microenvironment |
-
1999
- 1999-09-08 CN CNB991175891A patent/CN1134537C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CN1287166A (en) | 2001-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106659742B (en) | Genetically modified mesenchymal stem cells expressing immune response-stimulating cytokines to attract and/or activate immune cells | |
| CN1286981C (en) | Combination adeno-associated virus of expression human CYP2J2 antigene and its preparation method | |
| RU2205029C2 (en) | Genetically modified cells and their application for prophylaxis or treatment of sicknesses | |
| CN1838962A (en) | Injection of bone marrow-derived cells and medium for angiogenesis | |
| CN1134537C (en) | Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population | |
| CN109097401A (en) | A kind of preparation method of recombinant vector CAR-CD244-antiPSMA | |
| US6093393A (en) | Methods for preparing and using clonogenic fibroblasts and transfected clonogenic fibroblasts | |
| CN113430167A (en) | Culture method for simultaneously amplifying gamma delta T and NK | |
| WO2020259151A1 (en) | Preparation method and application of ctl cell | |
| CN107699591A (en) | A kind of knockout PD 1 T cell preparation method and applications | |
| CN114480505B (en) | Mesenchymal stem cells and anti-inflammatory application thereof | |
| CN106167791B (en) | MSC-TNF alpha-AB stem cell and preparation method and application thereof | |
| CN112770768A (en) | Production method for viral vectors | |
| CN113621611B (en) | Marrow specific promoter and application thereof | |
| CN108220243A (en) | The T cell and application of a kind of multipotential stem cell and its differentiation | |
| CN104830907A (en) | A constructing method of a bone marrow stem cell expressing an osteogenic gene | |
| WO2023030489A1 (en) | Application of genetically modified oligodendrocyte progenitor cell in multiple sclerosis | |
| WO2022247848A1 (en) | Preparation method for and application of hair follicle mesenchymal stem cell | |
| CN1520312A (en) | T cell induced tissue repair and regeneration | |
| CN106267419B (en) | HIV immunologic purging device | |
| CN111548998B (en) | Long-life plasma cell secreting PD-1 antibody and preparation method and application thereof | |
| CN107345221A (en) | A kind of TGF β 1 modify the preparation method of umbilical cord mesenchymal stem cells and the application in rheumatism | |
| CN1225285C (en) | Gene therapeutics | |
| WO2004071443A2 (en) | Methods and compositions for modulating stem cells | |
| AU717974B2 (en) | Transduced human hematopoietic stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: He Qinghua Document name: Notification of registration; notice of patent for invention |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: ZHAO CHUNHUA Free format text: FORMER OWNER: HE QINGHUA Effective date: 20070622 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20070622 Address after: 100005 No. three, No. 5, Beijing, Dongdan Patentee after: Zhao Chunhua Address before: 266071, Huaxia Bank, 11 floor, Galaxy Building, 29 Shandong Road, Shandong, Qingdao Patentee before: He Qinghua |
|
| ASS | Succession or assignment of patent right |
Owner name: JIANGSU LINGHANG STEM CELL REGENERATIVE MEDICINE E Free format text: FORMER OWNER: ZHAO CHUNHUA Effective date: 20100122 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20100122 Address after: Room 42, No. 610, Guangzhou Road, Nantong Development Zone, Jiangsu, China Patentee after: Jiangsu pilot stem cell regeneration medical engineering Co Ltd Address before: No. three, No. 5, Beijing, Dongdan Patentee before: Zhao Chunhua |
|
| C56 | Change in the name or address of the patentee | ||
| CP03 | Change of name, title or address |
Address after: 226009 Jiangsu Nantong economic and Technological Development Zone and Xing Road No. 176 Patentee after: GENESIS STEMCELL REGENERATIVE MEDICINE ENGINEERING CO., LTD. Address before: 610 room 42, 226000 Guangzhou Road, Nantong Development Zone, Jiangsu, China Patentee before: Jiangsu pilot stem cell regeneration medical engineering Co Ltd |
|
| CX01 | Expiry of patent term |
Granted publication date: 20040114 |
|
| CX01 | Expiry of patent term |