Slingshot-1L磷酸酶在人骨髓间充质干细胞成心肌诱导分化中的作用研究
摘要
本研究以体外培养的hMSCs为对象,联合应用密度梯度离心法,贴壁筛选法和单克隆培养法分离培养出高纯度的hMSCs,在体外可以传代培养和大量扩增,细胞在12代前生物学特性稳定,仍保持未分化状态,可作为后续实验的细胞来源。
应用5-Aza对hMSCs进行诱导,分别从形态特征、超微结构、mRNA水平、蛋白质水平以及电生理特性上对诱导后的hMSCs进行联合鉴定,证明诱导后的细胞已经成功向心肌细胞分化,即成功建立了体外hMSCs成心肌诱导体系。
经检测,5-Aza诱导后的hMSCs向心肌细胞分化的过程中Slingshot-1L(SSH-1L)的表达量呈一定的上升趋势,而SSH特异性底物Cofilin的磷酸化水平在诱导后呈明显的下降趋势,提示在hMSCs向心肌细胞分化的过程中SSH-1L可能被激活且对调控hMSCs向心肌细胞分化具有重要的作用。
本研究应用靶向SSH-1L基因的逆转录病毒表达载体pLNCX-SSH-1L转染至hMSCs,成功建立了过表达SSH-1L基因的hMSCs稳定转染细胞系,并研究SSH-1L在hMSCs成心肌诱导分化中的作用。结果显示,与对照组相比,SSH-1L基因转染组的hMSCs向心肌细胞的分化能力明显增强,包括从细胞形态,微丝骨架和电生理特性上加速hMSCs向心肌细胞的变化,上调心肌相关基因及心肌相关蛋白的表达水平。说明SSH-1L对于hMSCs向心肌细胞的分化具有明显的促进作用。
综上所述,本研究从不同角度深入地探讨了SSH-1L在骨髓间充质干细胞成心肌诱导分化中的作用。为进一步明确hMSCs向心肌细胞分化的机制提供有力的实验依据,指明了新的研究方向。
Adult cardiomyocytes(CMs) belong to permanent cells with limited ability of regeneration. Therefore, people are trying to seach newer ways to make the infracted cardiomyocytes to regenerate and promote revascularization.Recently, stem cellular transplantation has the possibility to replace infarctial cardiomyocytes and to restore function. So it becomes one of the hottest reach fields in rencent years.
Mesenchymal stem cells(MSCs)are the stem cells derived from mesoderm, possessing great multi-potent differentiation ability. MSCs can differentiate into many different kinds of cells under different environments.But the mechanisms of MSCs differentiated into cardiomyocytes are still not very clear . As reported, cytoskeleton can play an important role in the process of MSCs differentiation.
Slingshot(SSH) is a specific cofilin phosphatase. It can activate cofilin, inhibit the polymerization of actin filament,as a result, it participates in the rearrangement of cytoskeleton. This indicate that SSH may educe important effect in the process of MSCs’differentiation. Therefore, the stable transfected hMSCs cell line was established to study the effect of SSH phosphatase on hMSCs differentiation into cardiomyocytes. It may provide helpful theory support and laboratory basis for further study. A series of experiments were designed to investigate the effect of SSH-1Lphosphatase on hMSCs differentiation into cardiomyocytes.
Reslults:
1、A standardized platform has been established for hMSCs and the hMSCs maintaining stable characteristics, providing an enough cell source for further study.
2、We determined the proper inducing concentration of 5-Aza is 10μM by cell proliferation detection and observed the morphology of hMSCs differentiate into CMs.
3、We hint that 5-Aza can successfully induce hMSCs differentiating into CMs.
4、The retroviral vector pLNCX-SSH-1L was transfected into packaging cell PA317 by lipofectamine. The transfectants were selected and viral titers were 5×108 CFU /L,collect viral supernatant to infect hMSCs, abtain the monoclones overexpressing SSH-1L.
5、We carryed out a series of detections by Immunocytochemical staining, immuno- fluorescence, RT-PCR and Western blot to confirm that the stable transfected hMSCs cell line over-expression Slingshot mediated by retrovirus was established successfully, providing an enough cell source for further study on the effect of SSH phosphatase on hMSCs differentiation into CMs.
6、Karyotype analysis showing that Cr group(hMSCs which were not transfected), Scr (hMSCs which were transfected with vacant vector) and SSH group(hMSCs which were transfected with vector encoding SSH-1L) all maintained stable and normal diploid nucleus type without transform.
7、During hMSCs differentiate into CMs induced by 5–Aza for four weeks , their morphology change was observed .Compared to the Cr group and Scr group,SSH group hMSCs showed multiple branches after one week, connecting with adjoining cells forming myotube-like structures after 2~3weeks and the myotube-like structures increased strikingly after 3~4weeks.On the basis of these experiments,we conclude that SSH group hMSCs are more powerful in differentiating into CMs than the other two control groups.
8、We detected the microfilament changes during hMSCs differentiate into CMs induced by 5– Aza after four weeks. Cr, Scr and SSH group hMSCs’microfilament are all like long straight fiber bundle along the cell’s major axis with regular arrangement without induced.After induced by 5– Aza for one week, Cr group and Scr group hMSCs’microfilament basically along the cell’s major axis but with irregular arrangement,while SSH group hMSCs’microfilament spread to all directions. After induced for 2~3weeks, Cr group and Scr group hMSCs’microfilament spread to all directions, while SSH group hMSCs’microfilament join to one another and the actin gather together forming like intercalated disk structures. After induced for 3~4weeks, Cr group and Scr group hMSCs’microfilament join to one another forming like myotube-like structures, while SSH group hMSCs’actin gather together in microfilament junctions and forming like intercalated disk structures increase dramatically. On the basis of these experiments,we draw the conclusion that SSH-1Lplay an important role in hMSCs differentiation into CMs through regulating the rearrangement of cytoskeleton.
9、We then measured the hMSCs’action potential by current clamp and find that the hMSCs which were not induced by 5-Aza not exhibit action potential,while,those hMSCs after induced exhibit weak action potential and the action potential of SSH group is stronger than the other two control groups.
10、RT-PCR analysis of mRNA expression for cardiac-specific genes in hMSCs shows that the genes can’t be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, hMSCs express GATA4 and cTnT, which are early phase makers of cardimyogenic lineage.While SSH group hMSCs began to expressβ-MHC andα-sarcomeric actin which are late markers of cardiac lineage. After induced by 5– Aza for two weeks,Cr and Scr group hMSCs began to expressβ-MHC andα-sarcomeric actin.The mRNA expression level for cardiac-specific genes increased along with the elongation of induced time and the cardiac-specific gene expression level is much higher in SSH group hMSCs than in the other two control groups.
11、Western blot analysis showed that the expression of cardiac-specific protein can’t be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, SSH group hMSCs began to express Desmin, known to be early markers of myogenic differentiation. After induced for two weeks, Cr and Scr group began to express Desmin and then SSH group hMSCs begin to express of cardiac-specific protein cTnⅠandα-sarcomeric actin. While,Cr and Scr group expressed cTnⅠandα-sarcomeric actin until induced for three weeks. The cardiac-specific protein expression increased with the elongation of induced time and the SSH group hMSCs protein expression level is much higher than the other two control groups.
In short, this study systemically elucidate that 5-Aza can induce the differentiation of hMSCs into CMs in vitro. We confirmed that SSH-1L can efficiently promote MSCs’differentiation into CMs through regulating the rearrangement of cytoskeleton for the first time. Our work offers helpful theory support and laboratory basis for further investigate on the mechanisms of stem cells differentiate into CMs.
应用5-Aza对hMSCs进行诱导,分别从形态特征、超微结构、mRNA水平、蛋白质水平以及电生理特性上对诱导后的hMSCs进行联合鉴定,证明诱导后的细胞已经成功向心肌细胞分化,即成功建立了体外hMSCs成心肌诱导体系。
经检测,5-Aza诱导后的hMSCs向心肌细胞分化的过程中Slingshot-1L(SSH-1L)的表达量呈一定的上升趋势,而SSH特异性底物Cofilin的磷酸化水平在诱导后呈明显的下降趋势,提示在hMSCs向心肌细胞分化的过程中SSH-1L可能被激活且对调控hMSCs向心肌细胞分化具有重要的作用。
本研究应用靶向SSH-1L基因的逆转录病毒表达载体pLNCX-SSH-1L转染至hMSCs,成功建立了过表达SSH-1L基因的hMSCs稳定转染细胞系,并研究SSH-1L在hMSCs成心肌诱导分化中的作用。结果显示,与对照组相比,SSH-1L基因转染组的hMSCs向心肌细胞的分化能力明显增强,包括从细胞形态,微丝骨架和电生理特性上加速hMSCs向心肌细胞的变化,上调心肌相关基因及心肌相关蛋白的表达水平。说明SSH-1L对于hMSCs向心肌细胞的分化具有明显的促进作用。
综上所述,本研究从不同角度深入地探讨了SSH-1L在骨髓间充质干细胞成心肌诱导分化中的作用。为进一步明确hMSCs向心肌细胞分化的机制提供有力的实验依据,指明了新的研究方向。
Adult cardiomyocytes(CMs) belong to permanent cells with limited ability of regeneration. Therefore, people are trying to seach newer ways to make the infracted cardiomyocytes to regenerate and promote revascularization.Recently, stem cellular transplantation has the possibility to replace infarctial cardiomyocytes and to restore function. So it becomes one of the hottest reach fields in rencent years.
Mesenchymal stem cells(MSCs)are the stem cells derived from mesoderm, possessing great multi-potent differentiation ability. MSCs can differentiate into many different kinds of cells under different environments.But the mechanisms of MSCs differentiated into cardiomyocytes are still not very clear . As reported, cytoskeleton can play an important role in the process of MSCs differentiation.
Slingshot(SSH) is a specific cofilin phosphatase. It can activate cofilin, inhibit the polymerization of actin filament,as a result, it participates in the rearrangement of cytoskeleton. This indicate that SSH may educe important effect in the process of MSCs’differentiation. Therefore, the stable transfected hMSCs cell line was established to study the effect of SSH phosphatase on hMSCs differentiation into cardiomyocytes. It may provide helpful theory support and laboratory basis for further study. A series of experiments were designed to investigate the effect of SSH-1Lphosphatase on hMSCs differentiation into cardiomyocytes.
Reslults:
1、A standardized platform has been established for hMSCs and the hMSCs maintaining stable characteristics, providing an enough cell source for further study.
2、We determined the proper inducing concentration of 5-Aza is 10μM by cell proliferation detection and observed the morphology of hMSCs differentiate into CMs.
3、We hint that 5-Aza can successfully induce hMSCs differentiating into CMs.
4、The retroviral vector pLNCX-SSH-1L was transfected into packaging cell PA317 by lipofectamine. The transfectants were selected and viral titers were 5×108 CFU /L,collect viral supernatant to infect hMSCs, abtain the monoclones overexpressing SSH-1L.
5、We carryed out a series of detections by Immunocytochemical staining, immuno- fluorescence, RT-PCR and Western blot to confirm that the stable transfected hMSCs cell line over-expression Slingshot mediated by retrovirus was established successfully, providing an enough cell source for further study on the effect of SSH phosphatase on hMSCs differentiation into CMs.
6、Karyotype analysis showing that Cr group(hMSCs which were not transfected), Scr (hMSCs which were transfected with vacant vector) and SSH group(hMSCs which were transfected with vector encoding SSH-1L) all maintained stable and normal diploid nucleus type without transform.
7、During hMSCs differentiate into CMs induced by 5–Aza for four weeks , their morphology change was observed .Compared to the Cr group and Scr group,SSH group hMSCs showed multiple branches after one week, connecting with adjoining cells forming myotube-like structures after 2~3weeks and the myotube-like structures increased strikingly after 3~4weeks.On the basis of these experiments,we conclude that SSH group hMSCs are more powerful in differentiating into CMs than the other two control groups.
8、We detected the microfilament changes during hMSCs differentiate into CMs induced by 5– Aza after four weeks. Cr, Scr and SSH group hMSCs’microfilament are all like long straight fiber bundle along the cell’s major axis with regular arrangement without induced.After induced by 5– Aza for one week, Cr group and Scr group hMSCs’microfilament basically along the cell’s major axis but with irregular arrangement,while SSH group hMSCs’microfilament spread to all directions. After induced for 2~3weeks, Cr group and Scr group hMSCs’microfilament spread to all directions, while SSH group hMSCs’microfilament join to one another and the actin gather together forming like intercalated disk structures. After induced for 3~4weeks, Cr group and Scr group hMSCs’microfilament join to one another forming like myotube-like structures, while SSH group hMSCs’actin gather together in microfilament junctions and forming like intercalated disk structures increase dramatically. On the basis of these experiments,we draw the conclusion that SSH-1Lplay an important role in hMSCs differentiation into CMs through regulating the rearrangement of cytoskeleton.
9、We then measured the hMSCs’action potential by current clamp and find that the hMSCs which were not induced by 5-Aza not exhibit action potential,while,those hMSCs after induced exhibit weak action potential and the action potential of SSH group is stronger than the other two control groups.
10、RT-PCR analysis of mRNA expression for cardiac-specific genes in hMSCs shows that the genes can’t be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, hMSCs express GATA4 and cTnT, which are early phase makers of cardimyogenic lineage.While SSH group hMSCs began to expressβ-MHC andα-sarcomeric actin which are late markers of cardiac lineage. After induced by 5– Aza for two weeks,Cr and Scr group hMSCs began to expressβ-MHC andα-sarcomeric actin.The mRNA expression level for cardiac-specific genes increased along with the elongation of induced time and the cardiac-specific gene expression level is much higher in SSH group hMSCs than in the other two control groups.
11、Western blot analysis showed that the expression of cardiac-specific protein can’t be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, SSH group hMSCs began to express Desmin, known to be early markers of myogenic differentiation. After induced for two weeks, Cr and Scr group began to express Desmin and then SSH group hMSCs begin to express of cardiac-specific protein cTnⅠandα-sarcomeric actin. While,Cr and Scr group expressed cTnⅠandα-sarcomeric actin until induced for three weeks. The cardiac-specific protein expression increased with the elongation of induced time and the SSH group hMSCs protein expression level is much higher than the other two control groups.
In short, this study systemically elucidate that 5-Aza can induce the differentiation of hMSCs into CMs in vitro. We confirmed that SSH-1L can efficiently promote MSCs’differentiation into CMs through regulating the rearrangement of cytoskeleton for the first time. Our work offers helpful theory support and laboratory basis for further investigate on the mechanisms of stem cells differentiate into CMs.
引文
[1]李玉林,叶诸榕,龚守良.分子病理学,人民卫生出版社,第一版,2002,90-91.
[2]Janes SM,Lowell S,Hutter C.Epidermal stem cells[J]..J Pathol,2002,197 (4): 479-483.
[3]Taguchi Y,Yamamoto M,Yamatoe T,et al.Interleukin-6-type cytokines stimulate mese- nchymal progenitor differentiation toward the osteoblastic lineage[J].Proc Assoc Am Physicians,1998,110(6):559-574
[4]Pilloni A,Bernard GW.The effect of hyaluronan on mouse intramem- branous osteogenesis in vitro[J].Cell Tissue Res,1998,294(2):323-333
[5]Riew KD,Wright NM,Cheng S,et al.Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model[J].Calcif Tissue Int,1998,63(4):357-360
[6]Pittenger MF,Mackay AM,Beck SC,Jaiswal RK,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284:143-147.
[7]Bruder SP,Jaiswal N,Haynesworth SE.Growth kinetics,self-renewal,and the osteog- enic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation[J].Cell Biochem, 1997, 64(2): 278 -294.
[8]S.Kawano,S.Shoji,S.Ichinose,et al.Characterization of Ca2 +signaling pathways in human mesenchymal stem cells[J].Cell Calium,2002,32(4):165-174.
[9]Bruder SP,Kurth AA,SheaM,et al.Bone regeneration by implantation of purified,culture expanded human mesenchymal stem cells[J].Orthop Res, 1998,16:155-162.
[10]Worster AA,Nixon AJ,Brower Toland BD,et al.Effect of transforming growth factorβ1 on chondrogenic differentiation of cultured equine mesenchymal stem cells[J].Am J Vet Res,2000,61(9):1003-1010.
[11]Worster AA,Brower Toland BD,Fortier LA,et al.Chondrocytic differentiation of mesenc- hymal stem cells sequentially exposed to transforming growth factorβ1 inmonolayer and insulin-like growth factor-1 in a three-dimension matrix[J].J Orthop Res,2001,19(4): 738-749.
[12]Tsumaki N,Tanaka K,Arikawa HE,et al.Role of CDMP-1 in skeletal morphogenesis: prom- otion of mesenchymal cell recruitment and chondrocyte differentiation[J].J Cell Biol, 1999, 144(1):161-173.
[13]Shakibaei M.Inhibition of chondrogenesis by integrin antibody in vitro[J].Exp Cell Res, 1998,240(1):95-106.
[14]Johnstone B,Hering TM,Caplan AI,et al.In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells[J].Exp Cell Res, 1998,238(1): 265- 272.
[15]Bosnakovski D,Mizuno M,Kim G,et al.Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells(MSCs)in different hydrogels:Influence of collagen type II extracellular matrix on MSC chondrogenesis[J].Biotechnol Bioeng,2006,29(5): 720-729.
[16]70.Yoo JU,Barthel TS,Nishimura K,et al.The chondrogenic potential of human bone marrow derived meshenchymal progenitor cells[J].Bone Joint Surg Am Vol,1998, 80: 1745-1757.
[17]Rosen ED,Walkey CJ,Puigserver P,et al.Transcriptional regulation of adipogenesis [J].Genes Dev,2000,14(11):1293-1307.
[18]Neubauer M,Hacker M, et al.Adipose tissue engineering based on mesenchymal stem cells and basic fibroblast growth factor in vitro[J].Tissue Eng,2005,11(11-12).1840-1851.
[19]Wang JS , Chedrawy E , et al . The coronary delivery of marrow stromal cells for myocardial regeneration :pathophysiologic and therapeutic implications[J] . J Thorac Cardiovasc Surg ,2001 ,122 (4) :699-705.
[20]Cahill KS , Pittenger MF ,Byrne BJ . Differentiation of human messenchymal stem cells to cardiomyocytes by coculture [J] . Circlution , 2002 ,106 [ Suppl ] :198.-121
[21]Wakatani S,Saito T,Caplan A.Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine[J].Muscle Nerve, 1995, 18:1417-1426.
[22]Makino S,Fukuda K,Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro[J].Clin Invest,1999,103:697-705.
[23]Tomita S,Li RK,Weisel RD,et al.Autologous transplantation of bone marrow cells improves damaged heart function[J].Circulation,1999,100(19 Suppl):Ⅱ247.
[24]Konieczny SF,Emerson CP.5-Aazacytidine induction of stable mesenchymalstem cell lineage from 10 T 1/2 cells:evidence of regulatory genes controlling determination [J].Cell, 1984,38(2):791-800.
[25]Polychronis Antonitsis , Elisavet Ioannidou-Papagiannaki , et aI.In vitro cardiomyogenic differentiation of adult human bone marrow mesenchymal stem cells. The role of 5-azacy- tidine[J] . Interactive CardioVascular and Thoracic Surgery , 2007,6(1): 593–597
[26]Krause DS,Theise ND,Collector MI,et al.Multi-organ,multi-lineage engraftment by a single bone marrow-derived stem cells[J].Cell,2001;105(3):369-377.
[27]Kotton DN,Ma BY,Cardoso WV,et al.Bone marrow-derived cells as progenitors of lungalveolar epithelium[J].Development,2001,128(24):5181-5188.
[28]Oswald J,Boxberger S,Jorgensen B,et al.Mesenchymal stem cells can be differentiated into endothelial cells in vitro[J].Stem Cells,2004,22(3):377-384.
[29]Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenc- hymal stem cells[J].Science.1999;284(5411):143-147.
[30]Movaghar B,Tiraih,i T,Mesbah-Namin SA.,et al.Transdifferentiation of bone marrow stromal cells into Schwann cell phenotype using progesterone as inducer[J].Brain Res.2008, ) ) (6):205-208
[31]Thowfeequ S,Myatt EJ,Tosh D,et al.Transdifferentiation in develop- mental biology, disease,and in therapy.Dev Dyn[J].2007,Dec;236(12): 3208-3217.
[32]Annabi B,Lee YT,Turcotte S,et al.Hypoxia promotes murin bone-marrow-derived stromal cell migration and tube formation[J].Stem Cells,2003,21(3):337-347.
[33]Lennon DP,Edmison JM,Caplan AI.Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension:effects on in vitro and in vivo osteochondrogenesis[J].J Cell Physiol,2001,187(3):345-355.
[34]Woodbury D ,Schwarz EJ,Prockop DJ,et al.2000.Adult rat and human bone marrow stromal cells differentiate into neurons[J].J Neurosci Res, 61(4):364-367
[35]Jiang Y,Jahagirdar BN,Reinhardt RL,et al.2002.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,418(6893):41-49
[36]Zhao LR,Duan WM,Reyes M,et al.Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats[J].Exp Neurol.2002,174(1):11-20.
[37]Oh SH,Muzzonigro TM,Bae SH,et al.Adult bone marrow derived cells trans- differentiating into insulin-producing cells for the treatment of type I diabetes[J].Lab Invest,2004,84(5):607-617.
[38]Gerecht Nir S ,Eldor L ,Itskovitz2Eldor J . Advances in human stem cell research [J ] . Clin Obstet Gynecol ,2003,46 (2) :2182230.
[39]Kashiwakura Y,Katoh Y,Tamayose K, et al . Isolation of bone marrow stromal cell derived smooth muscle cells by a human SM22 promoter [J ] . Circulation , 2003,107 (16) :207822081.
[40]Moson JM,Breitbart AS ,Barcia M, et al . Cartilage and bone regeneration using gene enchanced tissue engineering[J ] . Clin Orthop ,2000 , 379[ Suppl ] : s1712 s178.
[41]Dennis JE ,Charbord P. Origin and differentiation of human and murine stroma [J ] . StemCells ,2002 ,20 (3) :2052214.
[42]Preston SL ,Alison MR , Forbes SJ , et al . The new stem cell biology : something for everyone[J ] .Mol Pathol ,2003 ,56 (2) :86296.
[43]Nakamura T, Shiojima S , Hirai Y, et al . Temproal gene expression changes during adipogenesis in human mesenchymal stem cells[J ] .Biochem Biophys Res Commun , 2003 ,303 (1) :306-312.
[44]Surani MA. Reprogramming of genome function through epigenetic in heritance [J ] . Nature ,2001 ,414 (6859) :122-128.
[45]Poulson R ,Allison MR ,Cook T, et al .Bone marrow stem cells contribute to healing of the kindney[J ] . J Am Soc Nephrol ,2003 ,14 [ Suppl 1 ] :s482s54.
[46]Caplan AZ,Bruder SP.Mesenchymal stem cells building blocks for molecular medicine in the 21st century[J ] . Trends Mol Med ,2001 ,7 (6) :259-264.
[47]Ioannis D,Nagy AH,Myrtle YA,et al.Adult bone marrow-derived stem cells and the injured heart:just the beginning[J].Europ J Cardio-thoracic Surg, 2005, 28(2): 665-676.
[48]Wlx Siepe M, Heilman C, von Samson P, Menasche P, Beyersdorf F. Stem cell research and cell transplantation for myocardial regeneration [J]. Eur J Cardiothor Surg , 2005, 28(1):318–324.
[49]Morito T , Muneta1 T , Hara K, et al . Synovial fluid-derived mesenchymal stem cells increase after int ra-articular ligament injury in humans [J] . Rheumatology , 2008,47(3) : 1137-1143.
[50]Toma C, PittengerMF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adultmurine heart[ J ]. Circulation, 2002, 105: 93- 98.
[51]Donald O, Jan K, Stdano C, et al. Bone marrow cells regenerate infracted myocardium [ J ]. Nature, 2001, 410:701 - 705.
[52]Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic s tem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts[J ]. Nature 2004, 428 (6983):664-668
[53]Balsam BL, Wagers JA, Chris tensen JL, et al. Haematopoietic s tem cells adopt mature haematopoietic fates in ischaemic myocardium[J ].Nature 2004, 428(6983): 668-673
[54]Nygren JM, Jovinge S, Breitbach M, et al.Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fus ion, but not transdifferen- tiation. Nat Med 2004,10(5):494-501
[55]Kajs tura J, Rota M, Whang B, et al. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fus ion[J ]. Circ Res 2005,96(1): 127-137
[56]KoyanagiM, Brandes RP, Haendeler J , et al. Cell to cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes: a novelmechanism for cell fate changes [ J ]. Circ Res, 2005, 96: 1039 - 1041.
[57]刘永刚,郭静萱.骨髓干细胞心肌内移植治疗的研究进展[ J ].中国介入心脏病学杂志, 2004, 12 ( 5) : 316 -318.
[58]Tang YaoL iang, Zhao Qiang, Qin Xinyu, et al. Paracrine action enhances the effects of autologousmesenchymal stem cell transp lantation on vascular regeneration in rat model of myocardial infarction [ J ]. Ann Thorac Surg, 2005, 80 (1) : 229 - 236.
[59]GnecchiM, He H, L iang OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt modified mesenchymal stem cells[ J ]. NatMed, 2005, 11(4) : 367 - 368.
[60]Ayach BB, YoshimitsuM, Dawood F, et al. Stem cell factor receptor induces progenitor and natural killer cell2medi2 ated cardiac survival and repair after myocardial infarction [ J ]. Proc Natl Acad Sci USA, 2006, 103 ( 7 ) : 2304 - 2309.
[61]Orlic D , Kajstura J , Chimenti S , et al . Bone marrow cells regenerate infracted myocardium[J ] . Nature , 2001 ,410 (6829) :701 - 705.
[62]Fazel S, CiminiM, Chen L, et al. Cardiop rotective c-Kit + cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines[ J ]. J Clin Invest, 2006, 116 (7) : 1865 - 1877.
[63]Mazhari R, Hare JM. Mechanisms of action of mesenchymal stem cells in cardiac repair: potential influences on the cardiac stem cell niche [ J ]. Nat Clin Pract Cardiovasc Med, 2007, 4 ( Supp l 1) : S21 - 26.
[64] Strauer BE, Brehm M, Zeus T, et al. Repair of infracted myocardium by autologous intracoronary mononuclear bone marrow cell transp lantation in humans [ J ]. Circulation, 2002, 106: 1913 - 1918.
[65]Chen SL, Fang WW, Qian J . Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal s tem cells in patients with acute myocardial infarction. Chin Med J (Engl) 2004,117 (10):1443-1448
[66]Janssens S, Dubois C, Bogaert J , et al. Autologous bone marrow derived stem cell transfer in patients with ST segment elevation myocardial infarction: doubleblind, randomised controlled trial[ J ]. Lancet, 2006, 367 ( 9505) :113 - 121.
[67]Dai W, Hale SL, Martin BJ.Allogeneic mesenchymal s tem cell transplantation in pos tinfarcted rat myocardium: short- and long-term effects . [ J ].Circulation 2005,112(2):214-223
[68]Yoon YS, Park JS, Tkebuchava T, et al.Unexpected severe calcification after transplan- tation of bone marrow cells in acute myocardial infarction[J ]. Circulation 2004, 109(25): 3147-3154
[69]Vulliet PR, Greeley M, Halloran SM, et al.Intracoronary arterial injection of mesen- chymal stromal cells and microinfarction in dogs[J ].Lancet 2004,363 (9411) : 783-784
[70]Rubio D, Garcia-Cas tro J , Martin MC, et al. Spontaneous human adult stem cell trans formation. Cancer Res 2005,65(8):3035-3039
[71]Zhang M, Methot D, Poppa V, et al.Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death s trategies[J ] .J Mol Cell Cardiol 2001,33(5): 907-921
[72]Davidoff AM, Ng CY, Brown P, et al. Bone marrow-derived cells contribute to tumor neovasculature and, when modified to expres s an angiogenes is inhibitor, can res trict tumor growth in mice[J ]. Clin Cancer Res 2001,7(9):2870-2879
[73]Majd Zayzafoon,William E.Gathings,JAY M.Modeled Microgravity Inhibits Osteogenic Differentiation of Human Mesenchymal Stem Cells and Increases Adipogenesis[J]. Endocrinology,2004,145(5):2421-2432.
[74] Xiaoyan Song, Xiaoming Chen, Hideki Yamaguchi, et al. Initiation of cofilin activity in response to EGF is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells[J]. Journal of Cell Science2006, 119:2871-2881
[75]Bailly,M.,Connecting cell adhesion to the actin polymerization machinery: vinculin as the missing link [J] .Trends Cell Biol.2003 13(4):163-5.
[76]Pavlov D, Muhlrad A, et al. Actin filament severing by cofilin[J]. J Mol Biol, 2007,365(5): 1350-1358
[77]Huang TY, DerMardirossian C, et al. Cofilin phosphatases and regulation of actin dynamics[J]. Curr Opin Cell Biol, 2006,18(1): 26-31
[78]Nishida E, Maekawa S, et al. Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin[J]. Biochemistry, 1984,23(22): 5307-5313
[79]Nishida E. Opposite effects of cofilin and profilin from porcine brain on rate of exchange of actin-bound adenosine 5'-triphosphate[J]. Biochemistry, 1985,24(5): 1160-1164
[80]Chen H, Bernstein BW, et al. Regulating actin-filament dynamics in vivo[J]. Trends Biochem Sci, 2000,25(1): 19-23
[81]Maciver SK, , Hussey PJ. The ADF/cofilin family: actin-remodeling proteins[J]. Genome Biol, 2002,3(5): reviews3007.
[82]Theriot JA,Mitchison TJ.The three faces of profilin.[J].Cell,1993,75(5):835-838
[83]Niwa R,Nagata-Ohashi K,Takeichi M,et al.Control of actin reorganization by Slingshot,a family of phosphatases that dephosphorylate ADF /cofilin [J]. Cell, 2002,108(2):233-246.
[84]Ohta Y,Kousaka K,Nagata-Ohashi K,et al.Differential activities, subcellular distribution and tissue expression patterns of three members of Slingshot family phosphatases that dephosphorylate cofilin.[J].Genes Cells,2003,8(10):811-824.
[85]Barford D,Das A K,Egloff MP.The structure and mechanism of protein phosphatases: insights into catalysis and regulation[J].Annu Rev Biophys Biomol Struct,1998, 27: 133- 164.
[86]Yan Wang, Futoshi Shibasaki, Kensaku Mizuno.Calcium Signal-induced Cofilin Dephosphorylation Is Mediated bySlingshot via Calcineurin[J]. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2005,280(13) : 12683–12689
[87]Mitsuharu Endo, Kazumasa Ohashi, and Kensaku Mizuno.LIM Kinase and Slingshot Are Critical for Neurite Extension[J].THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2007,282(18) : 13692–13702
[88]Michiru Nishita, Yan Wang, Chinatsu Tomizawa, et al. Phosphoinositide 3-Kinase-mediated Activation of Cofilin Phosphatase Slingshot and Its Role for Insulin- induced Membrane Protrusion[J].THE JOURNAL OF BIOLOGICAL CHEMISTRY 2004, 279(8) : 7193–7198
[89]Zhexing Wen,1 Liang Han,1 James R. Bamburg, ,et al. BMP gradients steer nerve growth conesby a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofi lin [J].THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2007,178(1) :107-119
[90]Souichi Kurita, Emi Gunji, Kazumasa Ohashi and Kensaku Mizuno. Actin filaments- stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1[J].Genes to Cells, 2007 ,12:663–676
[91]Pittenger MF,Mackay AM,Beck SC,Jaiswal RK,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284:143-147.
[92]呼莹,王秋英,马丽.胰腺源间充质干细胞的分离与鉴定[J].中国医学科学院学报,2002,24(1):45-49.
[93]Condorelli G,Borello U,Angelis LD,et al.Cardiomyocytes induce endothelial cells to transdifferentiate into cardiac muscle:Implications for myocardium regeneration [J]. PNAS.2001,98(19):10733-10738.
[94]Zhang FB,Li L,Fang B,et al.Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells[J].Biochem Biophy Res Commun. 2005, 336: 784-792.
[95]Doris AT.Cellular cardiomyoplasty with autologous skeletal myoblasts for ischemic heart disease and heart failure[J].Cur Control Trials in Cardiovasc Med. 2001,2:208-211.
[96]Konieczny SF,Emerson CP.5-Aazacytidine induction of stable mesenchymal stem cell lineage:evidence of regulatory genes controlling determination [J].Cell. 1984,38:791-800.
[97]Liu Y,Song J,Liu WX,et al.Growth and differentiation of rat bone marrow stromal cells:does 5-azacytidine trigger their cardiomyogenic differentiation[J]. Cardiovas Rese. 2003, 5 (8):460-468.
[98]150.Devine SM,Cobbs C,Jennings M,et al.Mesenchymal stem cells distribute to a wide range of tissues following systemic in fusion into nonhuman primates[J]. Blood. 2003,101(8):2999-3001.
[99]Bayes-Gents A,Salido M,Sole Ristol F,et al.Host cell-derived cardiomyocytes in sex-mismatch cardiac allografts[J].Cardiovasc Res.2002,56(3):404-410.
[100]翟中和,王喜忠,丁明孝.细胞生物学[M].第一版.北京:高等教育出版社, 2000:414-416.
[101]Farah CS,Reinach FC.The troponin complex and regulation of muscle contraction [J].FASEB J.1995,9:755.
[102]Jin JP,Huang QQ,Yeh HIL,et al.Complete nucleotide sequence and structural organization of rat cardiac troponin T gene:A single gene generates embryonic and adult isoforms via developmentally regulated alternative splicing[J].J Mol Biol. 1992, 227: 1269-1276.
[103]Panteghini M.Present issues in the determination of troponins and other markers of cardiac damage[J].Clin Biochem.2000,33(3):161-166.
[104]Lawsom-Smith MJ,McGeachie JK.The identification of myogenic cells in skeletal muscle,with emphasis on the use of tritiated thymidine autoradiography and desmin antibodies [J].J Anal.1998,192(Pt 2):161-71.
[105]Nishita, M., Wang, Y., Tomizawa, C. et al. Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion [J].J. Biol. Chem,2004,279:7193-7198.
[106]Huang TY, DerMardirossian C, et al. Cofilin phosphatases and regulation of actin dynamics[J].Curr Opin Cell Biol, 2006,18(1): 26-31
[107]Wilson DR. Viral mediated gene transfer for cancer treatment [J].Curr Pharm Biotechnol, 2002,3 :151-156
[108]李菁华,陈东,刘颖. mIL-12重组逆转录病毒载体的构建及包装细胞系的建立[J].吉林大学学报(医学版) ,2006,32(2):254-256
[109]臧卫东,赵青赞,王天云等.大鼠前脑啡肽原基因逆转录病毒表达载体及产毒细胞系的建立.郑州大学学报(医学版) ,2006,41(1):122-124
[2]Janes SM,Lowell S,Hutter C.Epidermal stem cells[J]..J Pathol,2002,197 (4): 479-483.
[3]Taguchi Y,Yamamoto M,Yamatoe T,et al.Interleukin-6-type cytokines stimulate mese- nchymal progenitor differentiation toward the osteoblastic lineage[J].Proc Assoc Am Physicians,1998,110(6):559-574
[4]Pilloni A,Bernard GW.The effect of hyaluronan on mouse intramem- branous osteogenesis in vitro[J].Cell Tissue Res,1998,294(2):323-333
[5]Riew KD,Wright NM,Cheng S,et al.Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model[J].Calcif Tissue Int,1998,63(4):357-360
[6]Pittenger MF,Mackay AM,Beck SC,Jaiswal RK,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284:143-147.
[7]Bruder SP,Jaiswal N,Haynesworth SE.Growth kinetics,self-renewal,and the osteog- enic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation[J].Cell Biochem, 1997, 64(2): 278 -294.
[8]S.Kawano,S.Shoji,S.Ichinose,et al.Characterization of Ca2 +signaling pathways in human mesenchymal stem cells[J].Cell Calium,2002,32(4):165-174.
[9]Bruder SP,Kurth AA,SheaM,et al.Bone regeneration by implantation of purified,culture expanded human mesenchymal stem cells[J].Orthop Res, 1998,16:155-162.
[10]Worster AA,Nixon AJ,Brower Toland BD,et al.Effect of transforming growth factorβ1 on chondrogenic differentiation of cultured equine mesenchymal stem cells[J].Am J Vet Res,2000,61(9):1003-1010.
[11]Worster AA,Brower Toland BD,Fortier LA,et al.Chondrocytic differentiation of mesenc- hymal stem cells sequentially exposed to transforming growth factorβ1 inmonolayer and insulin-like growth factor-1 in a three-dimension matrix[J].J Orthop Res,2001,19(4): 738-749.
[12]Tsumaki N,Tanaka K,Arikawa HE,et al.Role of CDMP-1 in skeletal morphogenesis: prom- otion of mesenchymal cell recruitment and chondrocyte differentiation[J].J Cell Biol, 1999, 144(1):161-173.
[13]Shakibaei M.Inhibition of chondrogenesis by integrin antibody in vitro[J].Exp Cell Res, 1998,240(1):95-106.
[14]Johnstone B,Hering TM,Caplan AI,et al.In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells[J].Exp Cell Res, 1998,238(1): 265- 272.
[15]Bosnakovski D,Mizuno M,Kim G,et al.Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells(MSCs)in different hydrogels:Influence of collagen type II extracellular matrix on MSC chondrogenesis[J].Biotechnol Bioeng,2006,29(5): 720-729.
[16]70.Yoo JU,Barthel TS,Nishimura K,et al.The chondrogenic potential of human bone marrow derived meshenchymal progenitor cells[J].Bone Joint Surg Am Vol,1998, 80: 1745-1757.
[17]Rosen ED,Walkey CJ,Puigserver P,et al.Transcriptional regulation of adipogenesis [J].Genes Dev,2000,14(11):1293-1307.
[18]Neubauer M,Hacker M, et al.Adipose tissue engineering based on mesenchymal stem cells and basic fibroblast growth factor in vitro[J].Tissue Eng,2005,11(11-12).1840-1851.
[19]Wang JS , Chedrawy E , et al . The coronary delivery of marrow stromal cells for myocardial regeneration :pathophysiologic and therapeutic implications[J] . J Thorac Cardiovasc Surg ,2001 ,122 (4) :699-705.
[20]Cahill KS , Pittenger MF ,Byrne BJ . Differentiation of human messenchymal stem cells to cardiomyocytes by coculture [J] . Circlution , 2002 ,106 [ Suppl ] :198.-121
[21]Wakatani S,Saito T,Caplan A.Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine[J].Muscle Nerve, 1995, 18:1417-1426.
[22]Makino S,Fukuda K,Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro[J].Clin Invest,1999,103:697-705.
[23]Tomita S,Li RK,Weisel RD,et al.Autologous transplantation of bone marrow cells improves damaged heart function[J].Circulation,1999,100(19 Suppl):Ⅱ247.
[24]Konieczny SF,Emerson CP.5-Aazacytidine induction of stable mesenchymalstem cell lineage from 10 T 1/2 cells:evidence of regulatory genes controlling determination [J].Cell, 1984,38(2):791-800.
[25]Polychronis Antonitsis , Elisavet Ioannidou-Papagiannaki , et aI.In vitro cardiomyogenic differentiation of adult human bone marrow mesenchymal stem cells. The role of 5-azacy- tidine[J] . Interactive CardioVascular and Thoracic Surgery , 2007,6(1): 593–597
[26]Krause DS,Theise ND,Collector MI,et al.Multi-organ,multi-lineage engraftment by a single bone marrow-derived stem cells[J].Cell,2001;105(3):369-377.
[27]Kotton DN,Ma BY,Cardoso WV,et al.Bone marrow-derived cells as progenitors of lungalveolar epithelium[J].Development,2001,128(24):5181-5188.
[28]Oswald J,Boxberger S,Jorgensen B,et al.Mesenchymal stem cells can be differentiated into endothelial cells in vitro[J].Stem Cells,2004,22(3):377-384.
[29]Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenc- hymal stem cells[J].Science.1999;284(5411):143-147.
[30]Movaghar B,Tiraih,i T,Mesbah-Namin SA.,et al.Transdifferentiation of bone marrow stromal cells into Schwann cell phenotype using progesterone as inducer[J].Brain Res.2008, ) ) (6):205-208
[31]Thowfeequ S,Myatt EJ,Tosh D,et al.Transdifferentiation in develop- mental biology, disease,and in therapy.Dev Dyn[J].2007,Dec;236(12): 3208-3217.
[32]Annabi B,Lee YT,Turcotte S,et al.Hypoxia promotes murin bone-marrow-derived stromal cell migration and tube formation[J].Stem Cells,2003,21(3):337-347.
[33]Lennon DP,Edmison JM,Caplan AI.Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension:effects on in vitro and in vivo osteochondrogenesis[J].J Cell Physiol,2001,187(3):345-355.
[34]Woodbury D ,Schwarz EJ,Prockop DJ,et al.2000.Adult rat and human bone marrow stromal cells differentiate into neurons[J].J Neurosci Res, 61(4):364-367
[35]Jiang Y,Jahagirdar BN,Reinhardt RL,et al.2002.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,418(6893):41-49
[36]Zhao LR,Duan WM,Reyes M,et al.Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats[J].Exp Neurol.2002,174(1):11-20.
[37]Oh SH,Muzzonigro TM,Bae SH,et al.Adult bone marrow derived cells trans- differentiating into insulin-producing cells for the treatment of type I diabetes[J].Lab Invest,2004,84(5):607-617.
[38]Gerecht Nir S ,Eldor L ,Itskovitz2Eldor J . Advances in human stem cell research [J ] . Clin Obstet Gynecol ,2003,46 (2) :2182230.
[39]Kashiwakura Y,Katoh Y,Tamayose K, et al . Isolation of bone marrow stromal cell derived smooth muscle cells by a human SM22 promoter [J ] . Circulation , 2003,107 (16) :207822081.
[40]Moson JM,Breitbart AS ,Barcia M, et al . Cartilage and bone regeneration using gene enchanced tissue engineering[J ] . Clin Orthop ,2000 , 379[ Suppl ] : s1712 s178.
[41]Dennis JE ,Charbord P. Origin and differentiation of human and murine stroma [J ] . StemCells ,2002 ,20 (3) :2052214.
[42]Preston SL ,Alison MR , Forbes SJ , et al . The new stem cell biology : something for everyone[J ] .Mol Pathol ,2003 ,56 (2) :86296.
[43]Nakamura T, Shiojima S , Hirai Y, et al . Temproal gene expression changes during adipogenesis in human mesenchymal stem cells[J ] .Biochem Biophys Res Commun , 2003 ,303 (1) :306-312.
[44]Surani MA. Reprogramming of genome function through epigenetic in heritance [J ] . Nature ,2001 ,414 (6859) :122-128.
[45]Poulson R ,Allison MR ,Cook T, et al .Bone marrow stem cells contribute to healing of the kindney[J ] . J Am Soc Nephrol ,2003 ,14 [ Suppl 1 ] :s482s54.
[46]Caplan AZ,Bruder SP.Mesenchymal stem cells building blocks for molecular medicine in the 21st century[J ] . Trends Mol Med ,2001 ,7 (6) :259-264.
[47]Ioannis D,Nagy AH,Myrtle YA,et al.Adult bone marrow-derived stem cells and the injured heart:just the beginning[J].Europ J Cardio-thoracic Surg, 2005, 28(2): 665-676.
[48]Wlx Siepe M, Heilman C, von Samson P, Menasche P, Beyersdorf F. Stem cell research and cell transplantation for myocardial regeneration [J]. Eur J Cardiothor Surg , 2005, 28(1):318–324.
[49]Morito T , Muneta1 T , Hara K, et al . Synovial fluid-derived mesenchymal stem cells increase after int ra-articular ligament injury in humans [J] . Rheumatology , 2008,47(3) : 1137-1143.
[50]Toma C, PittengerMF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adultmurine heart[ J ]. Circulation, 2002, 105: 93- 98.
[51]Donald O, Jan K, Stdano C, et al. Bone marrow cells regenerate infracted myocardium [ J ]. Nature, 2001, 410:701 - 705.
[52]Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic s tem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts[J ]. Nature 2004, 428 (6983):664-668
[53]Balsam BL, Wagers JA, Chris tensen JL, et al. Haematopoietic s tem cells adopt mature haematopoietic fates in ischaemic myocardium[J ].Nature 2004, 428(6983): 668-673
[54]Nygren JM, Jovinge S, Breitbach M, et al.Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fus ion, but not transdifferen- tiation. Nat Med 2004,10(5):494-501
[55]Kajs tura J, Rota M, Whang B, et al. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fus ion[J ]. Circ Res 2005,96(1): 127-137
[56]KoyanagiM, Brandes RP, Haendeler J , et al. Cell to cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes: a novelmechanism for cell fate changes [ J ]. Circ Res, 2005, 96: 1039 - 1041.
[57]刘永刚,郭静萱.骨髓干细胞心肌内移植治疗的研究进展[ J ].中国介入心脏病学杂志, 2004, 12 ( 5) : 316 -318.
[58]Tang YaoL iang, Zhao Qiang, Qin Xinyu, et al. Paracrine action enhances the effects of autologousmesenchymal stem cell transp lantation on vascular regeneration in rat model of myocardial infarction [ J ]. Ann Thorac Surg, 2005, 80 (1) : 229 - 236.
[59]GnecchiM, He H, L iang OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt modified mesenchymal stem cells[ J ]. NatMed, 2005, 11(4) : 367 - 368.
[60]Ayach BB, YoshimitsuM, Dawood F, et al. Stem cell factor receptor induces progenitor and natural killer cell2medi2 ated cardiac survival and repair after myocardial infarction [ J ]. Proc Natl Acad Sci USA, 2006, 103 ( 7 ) : 2304 - 2309.
[61]Orlic D , Kajstura J , Chimenti S , et al . Bone marrow cells regenerate infracted myocardium[J ] . Nature , 2001 ,410 (6829) :701 - 705.
[62]Fazel S, CiminiM, Chen L, et al. Cardiop rotective c-Kit + cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines[ J ]. J Clin Invest, 2006, 116 (7) : 1865 - 1877.
[63]Mazhari R, Hare JM. Mechanisms of action of mesenchymal stem cells in cardiac repair: potential influences on the cardiac stem cell niche [ J ]. Nat Clin Pract Cardiovasc Med, 2007, 4 ( Supp l 1) : S21 - 26.
[64] Strauer BE, Brehm M, Zeus T, et al. Repair of infracted myocardium by autologous intracoronary mononuclear bone marrow cell transp lantation in humans [ J ]. Circulation, 2002, 106: 1913 - 1918.
[65]Chen SL, Fang WW, Qian J . Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal s tem cells in patients with acute myocardial infarction. Chin Med J (Engl) 2004,117 (10):1443-1448
[66]Janssens S, Dubois C, Bogaert J , et al. Autologous bone marrow derived stem cell transfer in patients with ST segment elevation myocardial infarction: doubleblind, randomised controlled trial[ J ]. Lancet, 2006, 367 ( 9505) :113 - 121.
[67]Dai W, Hale SL, Martin BJ.Allogeneic mesenchymal s tem cell transplantation in pos tinfarcted rat myocardium: short- and long-term effects . [ J ].Circulation 2005,112(2):214-223
[68]Yoon YS, Park JS, Tkebuchava T, et al.Unexpected severe calcification after transplan- tation of bone marrow cells in acute myocardial infarction[J ]. Circulation 2004, 109(25): 3147-3154
[69]Vulliet PR, Greeley M, Halloran SM, et al.Intracoronary arterial injection of mesen- chymal stromal cells and microinfarction in dogs[J ].Lancet 2004,363 (9411) : 783-784
[70]Rubio D, Garcia-Cas tro J , Martin MC, et al. Spontaneous human adult stem cell trans formation. Cancer Res 2005,65(8):3035-3039
[71]Zhang M, Methot D, Poppa V, et al.Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death s trategies[J ] .J Mol Cell Cardiol 2001,33(5): 907-921
[72]Davidoff AM, Ng CY, Brown P, et al. Bone marrow-derived cells contribute to tumor neovasculature and, when modified to expres s an angiogenes is inhibitor, can res trict tumor growth in mice[J ]. Clin Cancer Res 2001,7(9):2870-2879
[73]Majd Zayzafoon,William E.Gathings,JAY M.Modeled Microgravity Inhibits Osteogenic Differentiation of Human Mesenchymal Stem Cells and Increases Adipogenesis[J]. Endocrinology,2004,145(5):2421-2432.
[74] Xiaoyan Song, Xiaoming Chen, Hideki Yamaguchi, et al. Initiation of cofilin activity in response to EGF is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells[J]. Journal of Cell Science2006, 119:2871-2881
[75]Bailly,M.,Connecting cell adhesion to the actin polymerization machinery: vinculin as the missing link [J] .Trends Cell Biol.2003 13(4):163-5.
[76]Pavlov D, Muhlrad A, et al. Actin filament severing by cofilin[J]. J Mol Biol, 2007,365(5): 1350-1358
[77]Huang TY, DerMardirossian C, et al. Cofilin phosphatases and regulation of actin dynamics[J]. Curr Opin Cell Biol, 2006,18(1): 26-31
[78]Nishida E, Maekawa S, et al. Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin[J]. Biochemistry, 1984,23(22): 5307-5313
[79]Nishida E. Opposite effects of cofilin and profilin from porcine brain on rate of exchange of actin-bound adenosine 5'-triphosphate[J]. Biochemistry, 1985,24(5): 1160-1164
[80]Chen H, Bernstein BW, et al. Regulating actin-filament dynamics in vivo[J]. Trends Biochem Sci, 2000,25(1): 19-23
[81]Maciver SK, , Hussey PJ. The ADF/cofilin family: actin-remodeling proteins[J]. Genome Biol, 2002,3(5): reviews3007.
[82]Theriot JA,Mitchison TJ.The three faces of profilin.[J].Cell,1993,75(5):835-838
[83]Niwa R,Nagata-Ohashi K,Takeichi M,et al.Control of actin reorganization by Slingshot,a family of phosphatases that dephosphorylate ADF /cofilin [J]. Cell, 2002,108(2):233-246.
[84]Ohta Y,Kousaka K,Nagata-Ohashi K,et al.Differential activities, subcellular distribution and tissue expression patterns of three members of Slingshot family phosphatases that dephosphorylate cofilin.[J].Genes Cells,2003,8(10):811-824.
[85]Barford D,Das A K,Egloff MP.The structure and mechanism of protein phosphatases: insights into catalysis and regulation[J].Annu Rev Biophys Biomol Struct,1998, 27: 133- 164.
[86]Yan Wang, Futoshi Shibasaki, Kensaku Mizuno.Calcium Signal-induced Cofilin Dephosphorylation Is Mediated bySlingshot via Calcineurin[J]. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2005,280(13) : 12683–12689
[87]Mitsuharu Endo, Kazumasa Ohashi, and Kensaku Mizuno.LIM Kinase and Slingshot Are Critical for Neurite Extension[J].THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2007,282(18) : 13692–13702
[88]Michiru Nishita, Yan Wang, Chinatsu Tomizawa, et al. Phosphoinositide 3-Kinase-mediated Activation of Cofilin Phosphatase Slingshot and Its Role for Insulin- induced Membrane Protrusion[J].THE JOURNAL OF BIOLOGICAL CHEMISTRY 2004, 279(8) : 7193–7198
[89]Zhexing Wen,1 Liang Han,1 James R. Bamburg, ,et al. BMP gradients steer nerve growth conesby a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofi lin [J].THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2007,178(1) :107-119
[90]Souichi Kurita, Emi Gunji, Kazumasa Ohashi and Kensaku Mizuno. Actin filaments- stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1[J].Genes to Cells, 2007 ,12:663–676
[91]Pittenger MF,Mackay AM,Beck SC,Jaiswal RK,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284:143-147.
[92]呼莹,王秋英,马丽.胰腺源间充质干细胞的分离与鉴定[J].中国医学科学院学报,2002,24(1):45-49.
[93]Condorelli G,Borello U,Angelis LD,et al.Cardiomyocytes induce endothelial cells to transdifferentiate into cardiac muscle:Implications for myocardium regeneration [J]. PNAS.2001,98(19):10733-10738.
[94]Zhang FB,Li L,Fang B,et al.Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells[J].Biochem Biophy Res Commun. 2005, 336: 784-792.
[95]Doris AT.Cellular cardiomyoplasty with autologous skeletal myoblasts for ischemic heart disease and heart failure[J].Cur Control Trials in Cardiovasc Med. 2001,2:208-211.
[96]Konieczny SF,Emerson CP.5-Aazacytidine induction of stable mesenchymal stem cell lineage:evidence of regulatory genes controlling determination [J].Cell. 1984,38:791-800.
[97]Liu Y,Song J,Liu WX,et al.Growth and differentiation of rat bone marrow stromal cells:does 5-azacytidine trigger their cardiomyogenic differentiation[J]. Cardiovas Rese. 2003, 5 (8):460-468.
[98]150.Devine SM,Cobbs C,Jennings M,et al.Mesenchymal stem cells distribute to a wide range of tissues following systemic in fusion into nonhuman primates[J]. Blood. 2003,101(8):2999-3001.
[99]Bayes-Gents A,Salido M,Sole Ristol F,et al.Host cell-derived cardiomyocytes in sex-mismatch cardiac allografts[J].Cardiovasc Res.2002,56(3):404-410.
[100]翟中和,王喜忠,丁明孝.细胞生物学[M].第一版.北京:高等教育出版社, 2000:414-416.
[101]Farah CS,Reinach FC.The troponin complex and regulation of muscle contraction [J].FASEB J.1995,9:755.
[102]Jin JP,Huang QQ,Yeh HIL,et al.Complete nucleotide sequence and structural organization of rat cardiac troponin T gene:A single gene generates embryonic and adult isoforms via developmentally regulated alternative splicing[J].J Mol Biol. 1992, 227: 1269-1276.
[103]Panteghini M.Present issues in the determination of troponins and other markers of cardiac damage[J].Clin Biochem.2000,33(3):161-166.
[104]Lawsom-Smith MJ,McGeachie JK.The identification of myogenic cells in skeletal muscle,with emphasis on the use of tritiated thymidine autoradiography and desmin antibodies [J].J Anal.1998,192(Pt 2):161-71.
[105]Nishita, M., Wang, Y., Tomizawa, C. et al. Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion [J].J. Biol. Chem,2004,279:7193-7198.
[106]Huang TY, DerMardirossian C, et al. Cofilin phosphatases and regulation of actin dynamics[J].Curr Opin Cell Biol, 2006,18(1): 26-31
[107]Wilson DR. Viral mediated gene transfer for cancer treatment [J].Curr Pharm Biotechnol, 2002,3 :151-156
[108]李菁华,陈东,刘颖. mIL-12重组逆转录病毒载体的构建及包装细胞系的建立[J].吉林大学学报(医学版) ,2006,32(2):254-256
[109]臧卫东,赵青赞,王天云等.大鼠前脑啡肽原基因逆转录病毒表达载体及产毒细胞系的建立.郑州大学学报(医学版) ,2006,41(1):122-124