大鼠骨髓间充质干细胞体外定向诱导分化为心肌样细胞的实验研究
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摘要
目的:探讨大鼠自体骨髓间充质干细胞在体外分离、培养方法,观察骨髓间充质干细胞的生物学特性;探讨对培养的骨髓间充质干细胞进行体外药物定向诱导,使其分化为心肌样细胞的方法及心肌样细胞的鉴定方法。
方法:共选取wistar雄性大鼠15只,体重180±20g,分为两组,一组7只,用Percoll分离培养法分离、培养;另一组8只,用全骨髓培养法分离、培养。15只大鼠均腹腔内注射戊巴比妥钠(30 mg/kg)麻醉,无菌条件下断离股骨,一组7只用PBS缓沖洗股骨干及干骺端,然后用Percoll液(密度为1.073g/ml)进行密度梯度离心后,取分界面的浑浊层,.用DMEM洗细胞2遍,接种到一次性塑料培养瓶中培养;另一组8只用DMEM培养液冲洗股骨干及干骺端,接种于培养瓶中,贴壁分离出骨髓间充质干细胞。将骨髓间充质干细胞混入培养体系(DMEM+15%特等优质胎牛血清+青霉素、链霉素各100 μg/ml;5%CO_2,37℃),扩增。待原代培养的细胞增殖到80%时,用0.25%牛胰蛋白酶消化细胞使之成为单细胞悬液,进行传代培养。
取生长良好的第三代骨髓间充质干细胞,胰酶消化后加入5-氮胞苷5μmol/L作用24小时,按正常培养条件培养,细胞长满培养瓶时(约5-6d)1:2.5传代,传代后进行同样的诱导、培养,倒置显微镜观察各期细胞形态的变化和生长发育情况。利用免疫荧光术、透射电镜等技术对诱导后的细胞进行鉴定。
结果:全骨髓培养法较Percoll密度梯度离心法培养的骨髓间充质干细胞纯
山东大学硕士学位论文
度高,细胞增殖能力强,在体外培养时细胞均一性较好,方法简单易行;spmol/L
的5一氮胞昔为诱导骨髓间充质干细胞向心肌样细胞分化较为适合的浓度;免疫
荧光可见a一Aetin,eardiae troponinl(eTnl)阳性细胞,透射电镜观察到部分细胞形成
肌丝样结构,细胞中可见到丰富的线粒体和糖原颗粒。
结论:1全骨髓培养法是一种简便易行、有效分离骨髓间充质干细胞的方法;
2 spmol/L的5一氮胞营为诱导骨髓间充质干细胞向心肌样细胞分化较为适合
的浓度;3骨髓间充质干细胞具有多能分化特性,体外培养的骨髓间充质干细胞
可在5一氮胞昔诱导下具有心肌细胞结构特点的心肌样细胞分化,为心肌细胞移
植打下坚实的细胞基础。
关键词:骨髓间充质干细胞、心肌细胞、全骨髓培养法、细胞分化、
5一氮胞普、免疫荧光术
Objective: To investigate the method of rat mesenchymal stem cells separated and cultured in vitro and to observe the biologic characteristics;To investigate the method by which rat mesenchymal stem cells can be induced into cardiomyocytes and the method which can identified them.Method: 15 Wistar rats,male,weighting 180±20g were separated into two groups randomly.One group is 7 rats,another is 8 rats.All of the rats were anesthetized with pentobarbital(30mg/kg body weight)by anintraperitoneal injection.Then the rats were come off the thighbones . The stem and epiphysis of thigbones of 7 rats were rinsed with PBS ,then rMSCs were isolated by Percoll separating medium(1.073g/ml) and cultured in culture bottles.Another 8 rats were rinsed with DMEM of 15% FBS,100ug/ml penicillin and 100ug/ml streptomycin.Finally,the rinsed water were inoculated in culture bottles and were cultured in vitro,observing rMSCs biologic characteristics.Then rMSCs were induced by 5-Aza 5umol/L for 24 hours.We identified that some of the induced rMSCs had differentiated into cardiomyocytes by means of transmission electron microscopy technique,immunofluorescent technique.Results: rMSCS isolated by the whole bone marrow culture had higher purity .better uniform and ability of proliferation in the process of culture in vitro than those of Percoll isolated method.5umol/L 5-Aza is an optimal concentration for rMSCs to differentite into cardiomyocytes.Some of the induced rMSCs were positive expression of a-Actin,cardiac troponinl (cTnl).Electron microscopy revealed
myofilament-like structure,rich glycogen granules and anumber of mitochondria.Conclusion: 1 The method of whole bone marrow culture is a well-referenced medium for density gradient centrifugation of rMSCs;2 The optimal conditions for rMSCs to differentiated into cardiomyocytes is 5umol/L 5-Aza;3 Mscs are mutipotential stem cells and they can be induced into cardiomyocytes by 5-Aza in vitro which have the characterestics of cardie myocytes in structure.
方法:共选取wistar雄性大鼠15只,体重180±20g,分为两组,一组7只,用Percoll分离培养法分离、培养;另一组8只,用全骨髓培养法分离、培养。15只大鼠均腹腔内注射戊巴比妥钠(30 mg/kg)麻醉,无菌条件下断离股骨,一组7只用PBS缓沖洗股骨干及干骺端,然后用Percoll液(密度为1.073g/ml)进行密度梯度离心后,取分界面的浑浊层,.用DMEM洗细胞2遍,接种到一次性塑料培养瓶中培养;另一组8只用DMEM培养液冲洗股骨干及干骺端,接种于培养瓶中,贴壁分离出骨髓间充质干细胞。将骨髓间充质干细胞混入培养体系(DMEM+15%特等优质胎牛血清+青霉素、链霉素各100 μg/ml;5%CO_2,37℃),扩增。待原代培养的细胞增殖到80%时,用0.25%牛胰蛋白酶消化细胞使之成为单细胞悬液,进行传代培养。
取生长良好的第三代骨髓间充质干细胞,胰酶消化后加入5-氮胞苷5μmol/L作用24小时,按正常培养条件培养,细胞长满培养瓶时(约5-6d)1:2.5传代,传代后进行同样的诱导、培养,倒置显微镜观察各期细胞形态的变化和生长发育情况。利用免疫荧光术、透射电镜等技术对诱导后的细胞进行鉴定。
结果:全骨髓培养法较Percoll密度梯度离心法培养的骨髓间充质干细胞纯
山东大学硕士学位论文
度高,细胞增殖能力强,在体外培养时细胞均一性较好,方法简单易行;spmol/L
的5一氮胞昔为诱导骨髓间充质干细胞向心肌样细胞分化较为适合的浓度;免疫
荧光可见a一Aetin,eardiae troponinl(eTnl)阳性细胞,透射电镜观察到部分细胞形成
肌丝样结构,细胞中可见到丰富的线粒体和糖原颗粒。
结论:1全骨髓培养法是一种简便易行、有效分离骨髓间充质干细胞的方法;
2 spmol/L的5一氮胞营为诱导骨髓间充质干细胞向心肌样细胞分化较为适合
的浓度;3骨髓间充质干细胞具有多能分化特性,体外培养的骨髓间充质干细胞
可在5一氮胞昔诱导下具有心肌细胞结构特点的心肌样细胞分化,为心肌细胞移
植打下坚实的细胞基础。
关键词:骨髓间充质干细胞、心肌细胞、全骨髓培养法、细胞分化、
5一氮胞普、免疫荧光术
Objective: To investigate the method of rat mesenchymal stem cells separated and cultured in vitro and to observe the biologic characteristics;To investigate the method by which rat mesenchymal stem cells can be induced into cardiomyocytes and the method which can identified them.Method: 15 Wistar rats,male,weighting 180±20g were separated into two groups randomly.One group is 7 rats,another is 8 rats.All of the rats were anesthetized with pentobarbital(30mg/kg body weight)by anintraperitoneal injection.Then the rats were come off the thighbones . The stem and epiphysis of thigbones of 7 rats were rinsed with PBS ,then rMSCs were isolated by Percoll separating medium(1.073g/ml) and cultured in culture bottles.Another 8 rats were rinsed with DMEM of 15% FBS,100ug/ml penicillin and 100ug/ml streptomycin.Finally,the rinsed water were inoculated in culture bottles and were cultured in vitro,observing rMSCs biologic characteristics.Then rMSCs were induced by 5-Aza 5umol/L for 24 hours.We identified that some of the induced rMSCs had differentiated into cardiomyocytes by means of transmission electron microscopy technique,immunofluorescent technique.Results: rMSCS isolated by the whole bone marrow culture had higher purity .better uniform and ability of proliferation in the process of culture in vitro than those of Percoll isolated method.5umol/L 5-Aza is an optimal concentration for rMSCs to differentite into cardiomyocytes.Some of the induced rMSCs were positive expression of a-Actin,cardiac troponinl (cTnl).Electron microscopy revealed
myofilament-like structure,rich glycogen granules and anumber of mitochondria.Conclusion: 1 The method of whole bone marrow culture is a well-referenced medium for density gradient centrifugation of rMSCs;2 The optimal conditions for rMSCs to differentiated into cardiomyocytes is 5umol/L 5-Aza;3 Mscs are mutipotential stem cells and they can be induced into cardiomyocytes by 5-Aza in vitro which have the characterestics of cardie myocytes in structure.
引文
1. Reida M,EL Oakley,MD et al. Myocyte transplantation for myocardial rpair:A few good cells can mend broken heart.Ann Thorac Surg,2001,71:1724-33.
2. Soonpaa MH, Koh GY, Klug MG, Field LJ. Science 1994 Apr 1;264 (5155): 98-101.
3. Bishop SP et al.Circ Res,1990;66:84~102.
4. Delcarpio JB et al.Circ Res,1991;69:1591~1600.
5. Tomita S, Li RK, Weisel RD. Circulation 1999 Nov 9;100(19 Suppl):II247-56.
6. Leor J,Patterson M,Quinones MJ .Circulation, 1996 Novl;94(9 suppl)II 332-11 336.
7. Li RK, Jia ZQ, Weisel RD, Mickle DA. Ann Thorac Surg 1996 Sep;62(3):654-60; discussion 660.
8. Marelli D et al.Cell Transplant, 1992; 1:383~390.
9. Ren-Ke Li, Richard D. Weisel, Donald A. G. J Thorac Cardiovasc Surg 2000; 119: 62-8.
10. Koh GY, Soonpaa MH, Klug MG, Field LJ. Am J Physiol 1993 May;264(5 Pt 2): H1727-33.
11. Li RK et al.Circ Res,1996;78: II179-181.
12. Orlic D,Kajsturn J,Chimenti S,et al.Bone marrow cells regenerate infracted myocardium. Nature,2001,410:701~705.
13. Blau HM,Webstr C.Isolation and characterization of human muscle cells.PNAS, 1981,78:5623-5627.
14. Wakitani S, Saito T, Caplan AI. Muscle Nerve 1995 Dec;18(12):1417-26 .
15. Shinji Tomita,Ren-Ke Li,Richard D.Circulation. 1999; 100: II -247.
16. Colter DC, Class R, DiGirolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 97:3213-3218, 2000.
17. S.E. Haynesworth, M.A. Baber, A.I. Caplan. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL- 1 alpha. J cell Physiol. 1996; 166:585.
18. Paul JS, Beberly TS. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, Stro-1. Blood 1991; 78(l):55-62.
19. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181:67-73, 1999.
20. Friedenstein AJ,Gorskkaja NN.Fibroblast precursors in normal and irradiated mouse hematopoiteic organs.Exp Hematol.l976;4(5):267-274.
21. 刘晓丹,郭子宽,李秀森等。人骨髓间充质干细胞分离与培养方法的建立。军事医学科学院院刊,2000;24(4):252-254.
22. Nakahara H,Dennis JE,Bruder SP et al.In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells.Exp Cell Res. 1991; 195 (2): 492-503.
23. Fortier LA,Nixon AJ,Williams J,et al.Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells.AM Jvet Res. 1998; 59 (9): 1182-1187.
24. Ghilzon R,Mc Culloch CA,Zohar R.Stromal mesenchymal progenitor cells in process xitation.Leuk Lymphoma.l999;32(3-4):211-221.
25. Zohar R, Sodek J, Mc Culloch CA,et al.Characteri-zation of stromal progenitor cells enriched by flow cytometry.Blood 1997;90(9):3471-3481.
26. Gronthos S,Grves SE,Ohta S,et al.The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors.Blood.1994;84:4164-4173.
27. Gordon EM,Stotzko M,Kundu RK,Han B,et al.Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-betal-von willebrand' factor fusion protein for retrovirus-mediated delivery of coagulation factor IX.Hum Gene Ther.1997 Jul 20;8(ll):1385-94.
28. Pittenger MF,Mackay AM,Beck SC,et l.Multilineage potentail of adult human mesenchymal stem cells.Science.l999;284(5411):143-147.
29. Manas KM, Mark AT, Joseph DM, et al. Phenotypic and functional comparison of culture of marrow-derived mesenchymal stem cells(MSCs) and stromal cells. Journal of Cellular Physiology 1998; 176:57-66.
30. Friedenstein AJ,Latzinik NV,Gorskaya YU,et al.Bone marrow stromal colony formation requires stimulation by haemopoictic cells.Bone Mineral. 1992; 36 (3): 225-32.
31. Lemienx ME,Eaves CJ.Identification of properties that can distinguish primitive populations of stromal-cell-responsive lympho-myeloid cells from cells that are stromal-cell-responsive but lymphoid-restricted and cells that have lympho-myeloid potential but are also capable of competitively repopulating myeloablatedrecipients.Blood.l996;88(5):1639-38.
32. Gordon MY,Lewis JL,Grand FH,et l.Phenotype and progeny of primitive adherent human hematopoietic progenitors.Leukemia.l996;10(8):1347-53.
33. Tomit S,Li RK,Weisel RD,et al.Autologous transplantation of bone marrow stem cells improves damaged heart function.Circulation.l999;100(suppl II ):247-256.
34. Phinney DG,Kopen G,Isaacson RL,et al.Plastic adherentstromal cells from the bone marrow of commonly used strains of inbred mice variations in yield, growth, and differentiation.J Cell Biochem.l999,72(4):570-585.
35. Konieczny SF,Emerson CP.5-Azacytidine induction of stable mesodermal stem cell linenges from 10Tl/2cells:evidence for regulatory genes controlling determination. Cell. 1984;38:791-800.
36. Malaval L,Modrowski D,Gupt AK,et al.Cellulaar expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell culture.J Cell Physiol. 1994;158:555-572.
37. Makino S,Fududa K,Miyoski S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest 1999;103:697-705.
38. 高峰,蔡振杰,俞世强等。大鼠骨髓间充质干细胞向心肌细胞的体外诱导分化。第四军医大学学报。2002;23(20)。
39. Peiffert SJaquet K,Helimeyer L,et al.Stepwise subunit interaction changes by mono and bisphosphorylation of cardiac troponin I. Biochemistry. 1998; 37: 13516-13525.
40. Toma C,Pittenger MF,Cahill KS,et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart, circulation. 2002; 105(l):93-8.
41. Malouf NH,Coleman WB,Grisham JW,et al.Adult-derived stem cells from the liver become myocytes in the heart in vivo.AM J Pathol.2001;158(6):1929-35.
42. Fukuda K. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artif Organs, 2001, 25: 187- 193.
43. Tomita S, Li RK, Weisel RD, et al. Autologous transPlantation of bon e marrow cells improves damaged heart function. Circulation, 1999, 100 (19 suppl): II247 -256.
44. Wang JS, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty: feasbility and potential clinical advantages. J Thorac Carduovasc Surg, 2000, 120:999-1005.
45. Wang JS, Shum-Tim D, Chedrawy E, et al. The coronary delivery of marrow stromal cells for myocardal regeneration: pathophysiologic and therapeutic implications. J Thorac Cardiovase Surg, 2001,122: 699-705.
46. Hakuno D, Fukuda K, Makino S, et al. Bone maroow-derived regeneraled cardiomyocytes (CMG Cells) express functional adrenergic and muscarinic receptors. Circulation, 2000, 105:380-386
47. Orlic D, Kajstura J, Chimenti s, et al. Bone marrow cells regenerate infarcted myocardium. Nature,2001, 410: 701-705.
48. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infareted heart, improving function and survival. Proc Natl Acad Sci USA, 2001, 98: 10344-10349.
49. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angioge ic ligands, and cytokines. Circulation, 2001, 104: 1046-1052.
50. Shake JG, Gruber PJ, Baumgartner WA, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg, 2002, 73: 1919-1926.
51. Strauer BE, Brehm M, Zeus T, et al. Intracoronary, human autologus stem cell transplantation for myocardial regeneration following myocard ial infaretion. Dtsch Med Wochenschr, 2001, 126: 932-938.
52. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med, 2000,6: 1282-1286.
53. Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation, 2002, 105: 93-98.
54. Saito T, Kuang JQ, Bittira B, et al. Xenotransplant cardiac chimera: inunune tolerance of adult stem cells.Ann Thorac Surg, 2002, 74: 19- 24.
2. Soonpaa MH, Koh GY, Klug MG, Field LJ. Science 1994 Apr 1;264 (5155): 98-101.
3. Bishop SP et al.Circ Res,1990;66:84~102.
4. Delcarpio JB et al.Circ Res,1991;69:1591~1600.
5. Tomita S, Li RK, Weisel RD. Circulation 1999 Nov 9;100(19 Suppl):II247-56.
6. Leor J,Patterson M,Quinones MJ .Circulation, 1996 Novl;94(9 suppl)II 332-11 336.
7. Li RK, Jia ZQ, Weisel RD, Mickle DA. Ann Thorac Surg 1996 Sep;62(3):654-60; discussion 660.
8. Marelli D et al.Cell Transplant, 1992; 1:383~390.
9. Ren-Ke Li, Richard D. Weisel, Donald A. G. J Thorac Cardiovasc Surg 2000; 119: 62-8.
10. Koh GY, Soonpaa MH, Klug MG, Field LJ. Am J Physiol 1993 May;264(5 Pt 2): H1727-33.
11. Li RK et al.Circ Res,1996;78: II179-181.
12. Orlic D,Kajsturn J,Chimenti S,et al.Bone marrow cells regenerate infracted myocardium. Nature,2001,410:701~705.
13. Blau HM,Webstr C.Isolation and characterization of human muscle cells.PNAS, 1981,78:5623-5627.
14. Wakitani S, Saito T, Caplan AI. Muscle Nerve 1995 Dec;18(12):1417-26 .
15. Shinji Tomita,Ren-Ke Li,Richard D.Circulation. 1999; 100: II -247.
16. Colter DC, Class R, DiGirolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 97:3213-3218, 2000.
17. S.E. Haynesworth, M.A. Baber, A.I. Caplan. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL- 1 alpha. J cell Physiol. 1996; 166:585.
18. Paul JS, Beberly TS. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, Stro-1. Blood 1991; 78(l):55-62.
19. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181:67-73, 1999.
20. Friedenstein AJ,Gorskkaja NN.Fibroblast precursors in normal and irradiated mouse hematopoiteic organs.Exp Hematol.l976;4(5):267-274.
21. 刘晓丹,郭子宽,李秀森等。人骨髓间充质干细胞分离与培养方法的建立。军事医学科学院院刊,2000;24(4):252-254.
22. Nakahara H,Dennis JE,Bruder SP et al.In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells.Exp Cell Res. 1991; 195 (2): 492-503.
23. Fortier LA,Nixon AJ,Williams J,et al.Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells.AM Jvet Res. 1998; 59 (9): 1182-1187.
24. Ghilzon R,Mc Culloch CA,Zohar R.Stromal mesenchymal progenitor cells in process xitation.Leuk Lymphoma.l999;32(3-4):211-221.
25. Zohar R, Sodek J, Mc Culloch CA,et al.Characteri-zation of stromal progenitor cells enriched by flow cytometry.Blood 1997;90(9):3471-3481.
26. Gronthos S,Grves SE,Ohta S,et al.The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors.Blood.1994;84:4164-4173.
27. Gordon EM,Stotzko M,Kundu RK,Han B,et al.Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-betal-von willebrand' factor fusion protein for retrovirus-mediated delivery of coagulation factor IX.Hum Gene Ther.1997 Jul 20;8(ll):1385-94.
28. Pittenger MF,Mackay AM,Beck SC,et l.Multilineage potentail of adult human mesenchymal stem cells.Science.l999;284(5411):143-147.
29. Manas KM, Mark AT, Joseph DM, et al. Phenotypic and functional comparison of culture of marrow-derived mesenchymal stem cells(MSCs) and stromal cells. Journal of Cellular Physiology 1998; 176:57-66.
30. Friedenstein AJ,Latzinik NV,Gorskaya YU,et al.Bone marrow stromal colony formation requires stimulation by haemopoictic cells.Bone Mineral. 1992; 36 (3): 225-32.
31. Lemienx ME,Eaves CJ.Identification of properties that can distinguish primitive populations of stromal-cell-responsive lympho-myeloid cells from cells that are stromal-cell-responsive but lymphoid-restricted and cells that have lympho-myeloid potential but are also capable of competitively repopulating myeloablatedrecipients.Blood.l996;88(5):1639-38.
32. Gordon MY,Lewis JL,Grand FH,et l.Phenotype and progeny of primitive adherent human hematopoietic progenitors.Leukemia.l996;10(8):1347-53.
33. Tomit S,Li RK,Weisel RD,et al.Autologous transplantation of bone marrow stem cells improves damaged heart function.Circulation.l999;100(suppl II ):247-256.
34. Phinney DG,Kopen G,Isaacson RL,et al.Plastic adherentstromal cells from the bone marrow of commonly used strains of inbred mice variations in yield, growth, and differentiation.J Cell Biochem.l999,72(4):570-585.
35. Konieczny SF,Emerson CP.5-Azacytidine induction of stable mesodermal stem cell linenges from 10Tl/2cells:evidence for regulatory genes controlling determination. Cell. 1984;38:791-800.
36. Malaval L,Modrowski D,Gupt AK,et al.Cellulaar expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell culture.J Cell Physiol. 1994;158:555-572.
37. Makino S,Fududa K,Miyoski S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest 1999;103:697-705.
38. 高峰,蔡振杰,俞世强等。大鼠骨髓间充质干细胞向心肌细胞的体外诱导分化。第四军医大学学报。2002;23(20)。
39. Peiffert SJaquet K,Helimeyer L,et al.Stepwise subunit interaction changes by mono and bisphosphorylation of cardiac troponin I. Biochemistry. 1998; 37: 13516-13525.
40. Toma C,Pittenger MF,Cahill KS,et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart, circulation. 2002; 105(l):93-8.
41. Malouf NH,Coleman WB,Grisham JW,et al.Adult-derived stem cells from the liver become myocytes in the heart in vivo.AM J Pathol.2001;158(6):1929-35.
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