CN1434119A - Establishment and use of adult stem cell restitution hemopoiesis method of mesenchyme stem cell phynotype - Google Patents
Establishment and use of adult stem cell restitution hemopoiesis method of mesenchyme stem cell phynotype Download PDFInfo
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Abstract
本发明公开了一种利用骨髓间充质干细胞表型的成体干细胞重建造血的方法。涉及了具有多系分化潜能的骨髓间充质源干细胞群的分离，联合G－CSF等细胞因子注射重建受超致死剂量Ce137照射后小鼠的造血功能。本发明在国际上首次利用骨髓间充质干细胞表型的成体干细胞群重建经超致死量照射骨髓衰竭动物的造血，并达到长期存活，建立间充质干细胞群联合G－CSF等细胞因子分化造血细胞的方法并可应用于造血障碍性疾病及多种造血细胞肿瘤疾病的治疗，从而为临床骨髓移植提供了新的骨髓移植途径。The invention discloses a method for reconstructing hematopoiesis using adult stem cells of bone marrow mesenchymal stem cell phenotype. It involves the isolation of bone marrow mesenchymal stem cell populations with multilineage differentiation potential, combined with the injection of G-CSF and other cytokines to rebuild the hematopoietic function of mice irradiated with a super-lethal dose of Ce 137 . For the first time in the world, the present invention uses adult stem cell populations of bone marrow mesenchymal stem cell phenotype to rebuild hematopoiesis in animals with bone marrow failure irradiated by superlethal doses, and achieves long-term survival. The cell method can be applied to the treatment of hematopoietic disorders and various hematopoietic tumor diseases, thus providing a new bone marrow transplantation approach for clinical bone marrow transplantation.
Description
The present invention relates to have the cytobiology fields such as multidirectional differentiation of the adult stem cell of mescenchymal stem cell phenotype, the medulla mesenchyma derived stem cell group who has a polyphyly differentiation potential by separation unites cytokine injections such as G-CSF and has set up a kind of method of rebuilding marrow hemopoiesis, thereby has verified in the body of adult stem cell polyphyly differentiation under specific environment and repaired pattern and propose the bone marrow transplantation New Policy from body.
At present, transplanting is clinically mobilized blood, umbilical cord blood transplantation based on marrow, periphery, and the simplified marrow transplanting immunological rejection problem of hemopoietic stem cell and periphery mobilize the problems such as hemopoietic stem cell quality and quantity of blood or umbilical cord blood transplantation all to await further solving and overcoming, along with the understanding to the adult stem cell differentiation mechanism is progressively goed deep into, we propose adult stem cell and have the inferior myeloid-lymphoid stem cell group of polyphyly differentiation potential in the tissue for being present in, and can induce to be divided into multiple histocyte under specific environment.Pastern bone marrow mesenchyme derived stem cell group of the present invention breaks up hemopoietic stem cell hematopoietic reconstitution autotransplantation pattern, from newborn infant, mouse and rabbit bone marrow, isolate the mescenchymal stem cell group in the fetal pancreas, prove that it has the feature of multipotential stem cell, carried out the interior transplantation experiments of body of mesenchymal stem cells MSCs thus.Application has the adult stem cell of mescenchymal stem cell phenotype and has successfully rebuild the hematopoiesis of the BALB/C mice that is subjected to the supralethal irradiation, and has identified the donor mescenchymal stem cell hematopoiesis source that rebuilds the acceptor mouse.The present invention is population of adult stem cells new purposes in bone marrow transplantation.
The objective of the invention is to utilize first in the world the population of adult stem cells of mesenchymal stem cells MSCs phenotype to rebuild the hematopoiesis of shining the marrow failure animal through supralethal, and reach long-term surviving, set up the mescenchymal stem cell group and unite the method for cytokine such as G-CSF differentiation hematopoietic cells and can be applicable to the hematopoietic disorders disease and multiple hematopoietic tumor treatment of diseases, thereby provide new bone marrow transplantation approach for clinical bone marrow transplantation.
The objective of the invention is (Fig. 1) by following method realization:
One, adopt liquid culture to separate, the amplification mesenchymal stem cells MSCs is as donorcells
(1) gets human or animal's marrow, counting cells, lymphocyte separation medium (Ficoll 1.077g/ml) separates mononuclearcell, centrifugal 1500 rev/mins, 20 minutes, get the above cell part of white ring, be resuspended in the RPMI1640 nutrient solution (GibcoBRL company) counting cells, with containing DMEM-LG/F12, MCDB-201,2%FCS (GibcoBRL company) nutrient solution is cultivated, and inoculum density is 1-2 * 10
6/ ml.Put 37 ℃, 5%CO
2Incubator is cultivated, and changes liquid after one day, discards non-adherent cell, and full dose was changed liquid in later per two days.To cultivating the back the 10th day, cell reaches 80% and merges, with 0.25% pancreatin (Sigma company) digestion, infusion.
The evaluation of donorcells: cell is placed the 3.5cm diameter plate of little slide, 37 ℃, saturated humidity, 5%CO
2Cultivate after 12 days that to take out air air-dry, Switzerland's dyeing, observation of cell form under the oily mirror.Inverted microscope is observed down, and most of cell is promptly adherent in 24 hours, shows as the inoblast sample, has cellular fories such as fusiformis, polygon.
Cell is fixed 2 hours with 2.5% glutaraldehyde, cleans through PBS, and 1% osmic acid is fixed 2 hours, the dehydration of acetone gradient, and through conventional Ep0812 embedding, ultramicrotome section after the polymerization.The dyeing of acetic acid uranium lead citrate is observed as seen based on fibroblast under the H-100 electron microscope, and characteristics are microscler, spindle cell, the nuclear heterochromatin is few, kernel is obvious, and rough surfaced endoplasmic reticulum, microtubule, the microfilament of expansion arranged in the endochylema, visible endoplasmic reticulum secreting outside microfilament.Organoid is few in a few cell endochylema, and endochylema is few, and kernel is obvious, may be stem cell.
Get each for cell, the digestion counting is inoculated in 24 orifice plates interior (1 * 10
4Cells/well), every other day get two porocytes and count, computation of mean values is drawn growth curve.In the logarithmic phase of cell growth, the mesenchymal stem cells MSCs doubling time is about 25 hours.The every biography generation of cell cell count increases by 2 times approximately.
(2) with cell dissociation, 70% cold ethanol is fixed 24 hours for 4 ℃, and 10mg/ml PI dyes (4 ℃, lucifuge 30 minutes), detects ModFIT software analysis result with flow cytometer (FACSVantage type).Cell cycle analysis finds that the cell above 80% all is in the G0/G1 phase.Utilize flow cytometer that culturing cell is carried out phenotype test CD34, HLA-DR is negative, and CD44, CD29, CD13 are then positive.
SA method proof mesenchymal stem cells MSCs I, III Collagen Type VI, the vWF factor expression positive.
(3) measure each telomerase activation, prove its Telomerase high expression level for cell.
(4) the interior polyphyly of external and body is induced differentiation, when cell reaches 80% fusion, with 5 * 10
3/ cm
2Be inoculated in 25cm
2The culture dish of culturing bottle and 6cm diameter, Osteoblast Differentiation system 10
-7The IMDM of M dexamethasone, 10mM β-phospho-glycerol, 0.05mM vitamins C and 10%FCS; The fat differentiated system contains 10
-6The IMDM of M dexamethasone, 0.5mM3-isobutyl-1-methylxanthine (IBMX), 0.1mM vitamins C and 10%FCS.Become cartilage differentiated system DMEM-HG, MCDB, 2%FCS, dexamethasone 10
-7M, TGF-β 10ng/ml.Can successful induced osteogenesis cell, chondroblast, adipocyte, Skeletal Muscle Cell, neurocyte transform.
(5) detection of external evoked differentiation, detected calcification brief summary, fat granule, alkaline phosphatase staining, sudan black dyeing, and osteocalcin, the isogenic expression of lipoprotein lipase respectively at the 4th day, the 8th day, the 12nd day and the 16th day, thus the skeletonization of identifying mescenchymal stem cell with become fatty differentiation potential.The alcian blue staining detects protein-polysaccharide, and the immunohistochemical methods method detects the II Collagen Type VI, and the RT-PCR method detects II Collagen Type VI genetic expression etc., thereby identifies that mescenchymal stem cell becomes the cartilage differentiation potential.
(6) extract culturing cell RNA and adopt the RT-PCR method to detect GATA-1 in the culturing cell, GATA-2, c-Myb, cKit, the expression of mRNA such as PU.1, all negative when proving to body injection, there is not hematopoietic stem/progenitor cells to pollute in the culture system.
Two, the experimental study of reconstitute hematopoiesis in the body
With 12 all acceptor BALB/C mice Ce
137Import 6 * 10 behind the full-body exposure 850rad
6/ 0.3ml/ is the mesenchymal stem cells MSCs after cultivating only, the 1st the sky behind the infusion cell, afternoon each intramuscular injection G-CSF.Observe the survival condition of mouse.
Mouse is looked into tail vein before infusion, meter total cellular score, smear, classification are respectively got 1 mouse weekly and got liver, spleen, femur after the transfusion cell, fixedly does the pathology section, and an other side femur is gone out marrow, counting karyocyte, smear, mensuration GM-CFU value.PCR method identifies that the hemocyte of rebuilding mouse is from donorcells.Defeated full marrow group peripheral blood karyocyte and bone marrow nucleated cell number and GM-CFU number after defeated the 1st, all be higher than single defeated mesenchymal stem cells MSCs group 2 weeks, but single defeated medulla mesenchyma groups of cells week blood karyocyte recovery is very fast, mouse survives, the 3rd week to the infusion, karyocyte in all blood or the marrow all obviously recovers than the 2nd week, and GM-CFU also obviously raises, and single defeated hematopoietic cell and D-hanks liquid group mouse survival death in 4-5 days.Prompting utilizes the population of adult stem cells of mesenchymal stem cells MSCs phenotype can rebuild the hematopoiesis of shining the marrow failure animal through supralethal, and reaches long-term surviving.The method that the mescenchymal stem cell group unites cytokine differentiation hematopoietic cells such as G-CSF can be applicable to hematopoietic disorders disease and multiple hematopoietic tumor treatment of diseases, for clinical bone marrow transplantation provides new bone marrow transplantation approach.
Three, the application examples with marrow adult stem cell reconstitute hematopoiesis of mescenchymal stem cell phenotype is done
The employing liquid culture is separated, and the amplification mesenchymal stem cells MSCs as donorcells, is got the BALB/C mice four limbs and removed muscular fascia, go out marrow, counting cells is with containing DMEM-LG/F12, MCDB-201,2%FCS (GibcoBRL company) nutrient solution is cultivated, and inoculum density is 1-2 * 10
6/ ml.Put 37 ℃, 5%CO
2Incubator is cultivated, and changes liquid after one day, discards non-adherent cell, and later per three and half amounts are changed liquid.To cultivating the back the 10th day, with the conventional digestion of 0.25% pancreatin (Sigma company).The evaluation of donorcells: cell shows as the inoblast sample, has cellular fories such as fusiformis, polygon.
Electron microscopic observation is as seen based on fibroblast, visible endoplasmic reticulum secreting outside microfilament.Organoid is few in a few cell endochylema, and endochylema is few, and kernel is obvious, may be stem cell.
Growth curve is measured: in the logarithmic phase of cell growth, the mesenchymal stem cells MSCs doubling time is about 25 hours.The every biography generation of cell cell count increases by 2 times approximately.
Cell cycle is detected: the cell above 80% all is in the G0/G1 phase.CD34, HLA-DR is negative, and CD44, CD29, CD13 are then positive.SA method proof mesenchymal stem cells MSCs I, III Collagen Type VI, the vWF factor expression positive.Measure each telomerase activation, prove its Telomerase high expression level for cell.
Polyphyly is induced differentiation in the external and body, can successful induced osteogenesis cell, chondroblast, adipocyte, Skeletal Muscle Cell, neurocyte transform.The RT-PCR method detects GATA-1 in the culturing cell, and GATA-2, c-Myb, cKit, PU.1mRNA express when injecting to body all negative, and proving does not have hematopoietic stem/progenitor cells to pollute in the culture system.
More than the medullary cell of proof cultivation is the adult stem cell with mescenchymal stem cell phenotype.Can be used as the donorcells of reconstitute hematopoiesis in the body.
In the mouse body, carry out the experiment of reconstitute hematopoiesis: 12 all acceptor BALB/C mice are divided into 5 groups, every group of 12 Ce
1371. 2. shine tail venoclysis 6 * 10 in back 4 hours without any processing after the normal mouse irradiation behind the full-body exposure 850rad
63. the only fresh full marrow of/0.3ml/ import 6 * 10
6/ 0.3ml/ only goes the hematopoietic cell of stroma cell 4. to import 6 * 10
6/ 0.3ml/ is the stroma cell after cultivating only, the 1st the sky behind the infusion cell, afternoon each intramuscular injection G-CSF.5. shine tail venoclysis 6 * 10 in back 4 hours
6/ 0.3ml/ only cultivates the 10th day mesenchymal stem cells MSCs.The 1st day upper and lower noon of injected in mice G-CSF respectively once behind the infusion cell.The careful nursing observed the survival condition of respectively organizing mouse.
Mouse is looked into tail vein before infusion, meter total cellular score, smear, classification, after the transfusion cell weekly each group get 1 mouse and get liver, spleen, femur, fixedly do the pathology section, an other side femur is gone out marrow, counts karyocyte, smear, mensuration GM-CFU value.Fail all blood karyocytes of full marrow group and bone marrow nucleated cell number and the GM-CFU number list that all was higher than in the 1st, 2 week after failing and fail marrow adult stem cell group, but list is failed all blood karyocyte recoveries of marrow adult stem cell group comparatively fast, mouse survives.In the 3rd week to the infusion, the karyocyte in all blood or the marrow all obviously recovers (Fig. 2,3) than the 2nd week, and GM-CFU also obviously raises, and single defeated hematopoietic cell and D-hanks liquid group mouse survival death in 4-5 days.Fig. 4 shows that single defeated adult stem cell group GM-CFU value is in beginning recovery second week and surpassing defeated full marrow group in the 3rd week.Fig. 5 shows, by increase the specifically sry gene of Y chromosome of PCR method, determines that the hematopoietic cell major part derives from donor in the mouse body of reconstitute hematopoiesis.Fig. 6 shows, spleen colony changing conditions in the hematopoietic reconstitution process, and in second week behind the infusion, adult stem cell infusion group spleen colony is obviously more than full bone marrow infusion group; In the 3rd week behind the infusion, both do not have significant difference.
Advantage of the present invention:
1,, proved mescenchymal stem cell group's versatility by research to mescenchymal stem cell source population of adult stem cells reconstitute hematopoiesis.
2, by research, prove that adult stem cell is to be present in the inferior myeloid-lymphoid stem cell group who has the polyphyly differentiation potential in the tissue, can induce to be divided into multiple histocyte under specific environment to mescenchymal stem cell source population of adult stem cells reconstitute hematopoiesis.
Caption Fig. 1: experiment flow 1: donor bone marrow from male mice 2: the adult stem cell 3 with mescenchymal stem cell phenotype: the adult stem cell 4 of tail vein injection mescenchymal stem cell phenotype: bone marrow cfu-gm measures 5: pathological examination Fig. 2: 1: full bone marrow infusion 2: the adult stem cell infusion 3 of mescenchymal stem cell phenotype: normal control Fig. 3: 1: full bone marrow infusion 2: the adult stem cell infusion 3 of mescenchymal stem cell phenotype: normal control Fig. 4: 1: full bone marrow infusion 2: adult stem cell infusion Fig. 5 of mescenchymal stem cell phenotype: 1: molecular weight marker 2: normal female mouse 3: normal male mouse 4: full bone marrow infusion 5: adult stem cell infusion Figure 61 of mescenchymal stem cell phenotype: second week 2 behind the cell infusion: the 3rd week behind the cell infusion. The right side is full bone marrow infusion; The left side is the adult stem cell infusion of mescenchymal stem cell phenotype
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009030092A1 (en) * | 2007-09-05 | 2009-03-12 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on a large scale, primary mesenchymal stem cells obtained by the method, the uses thereof |
| CN1778905B (en) * | 2004-11-22 | 2010-05-05 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| CN110468099A (en) * | 2019-07-19 | 2019-11-19 | 海门生原干细胞科技有限公司 | A kind of mesenchymal stem cell culture medium and its application |
| CN120939057A (en) * | 2025-08-15 | 2025-11-14 | 重庆国际免疫研究院 | Application of costal cartilage-derived stem cells and their combination with pluripotent progenitor cells, hematopoietic stem cells, or cyclosporine in the preparation of formulations for hematopoietic reconstitution. |
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2001
- 2001-09-19 CN CN01140511A patent/CN1434119A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778905B (en) * | 2004-11-22 | 2010-05-05 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| WO2009030092A1 (en) * | 2007-09-05 | 2009-03-12 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on a large scale, primary mesenchymal stem cells obtained by the method, the uses thereof |
| CN101821383B (en) * | 2007-09-05 | 2013-12-18 | 中国医学科学院基础医学研究所 | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on large scale, primary mesenchymal stem cells obtained by method, uses thereof |
| CN110468099A (en) * | 2019-07-19 | 2019-11-19 | 海门生原干细胞科技有限公司 | A kind of mesenchymal stem cell culture medium and its application |
| CN110468099B (en) * | 2019-07-19 | 2023-08-15 | 海门生原干细胞科技有限公司 | Bone marrow mesenchymal stem cell culture medium and application thereof |
| CN120939057A (en) * | 2025-08-15 | 2025-11-14 | 重庆国际免疫研究院 | Application of costal cartilage-derived stem cells and their combination with pluripotent progenitor cells, hematopoietic stem cells, or cyclosporine in the preparation of formulations for hematopoietic reconstitution. |
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