脐血间充质干细胞修复心肌的研究和干细胞临床应用
|
摘要
研究目的(1)探讨脐血间充质干细胞可否成功的在体外分离、培养、扩增并诱导成心肌样细胞；探讨脐血来源的间充质干细胞的分离鉴定,经体外扩增、纯化的方法以及定向分化成心肌样细胞的诱导剂5-氮胞苷(5-AZA)作用后的生物学特性；(2)观察脐血间充质干细胞与心肌细胞在体外共同培养,与脐血MSCs共同培养的心肌细胞是否具有抵抗缺氧引起的凋亡;(3)观察体外进行长期的培养和扩增脐血MSCs,以及对细胞的遗传物质有修饰作用的试剂5-AZA进行体外诱导后的脐血MSCs的生物安全性；(4)进行重组人粒细胞集落刺激因子(G-CSF)对进行骨髓动员,然后分离纯化其外周血单个核细胞,将单核细胞经心导管在梗死相关动脉内注射治疗的急性心肌梗死患者进行临床指标的观察。
方法与结果
第一部分：提取健康妊娠产妇分娩的胎儿脐带血,分离单个核细胞进行培养、传代,取第3代以后细胞,用流式细胞仪直接标记分析CD34、CD44、CD90细胞表面标志。用10μmmol/L的5-AZA诱导4周后,倒置显微镜下观察细胞呈类心肌细胞样改变、透视电镜观察：细胞核呈椭圆形,胞浆内可见成束的肌丝,肌丝内可见梭形密体、丰富的糖原颗粒、大量的线粒体、内质网发达。免疫荧光组化染色体可见：GATA4, cTnI表达阳性的细胞。并检测细胞β-肌球蛋白重链(β-MyHC)基因的表达。5-AZZ作用后细胞进行生长周期分析以及生长曲线描记。结果显示体外纯化扩增后的脐血间充质干细胞含有与骨髓来源的间充质干细胞相同的细胞表面标志,第三代以后细胞纯度较高,经5-AZA在体外诱导培养,可形成心肌样细胞,在诱导剂量作用后,基本的生物学特性未发生改变。
第二部分：将人的心肌细胞置于95%N2-5%CO2缺氧环境下培养造成模拟心肌缺血损伤模型。实验结果显示细胞凋亡与缺氧呈时间依赖性,将人的心肌细胞和人脐血MSCs在缺氧环境下共同培养,脐血MSCs具有抵抗缺氧引起的心肌细胞凋亡的能力。
第三部分：通过对体外长期培养、扩增的脐血MSCs细胞和经5-AZA诱导作用28天后的MSCs细胞的生长曲线和生长周期分析、细胞核型分析、端粒酶活性检测、细胞周期相关基因表达的检测以及细胞成瘤实验来评价试验用脐血MSCs是否发生恶性转化。结果证实：经离体培养、扩增脐血MSCs和经5-AZA诱导分化成心肌样细胞后不存在基因突变,不具有成瘤性。
第四部分：选择6例急性心肌梗死的病人,在移植前1周,应用干细胞动员剂,重组人粒细胞集落刺激因子(G-CSF)对进行骨髓动员,然后用细胞分离仪对外周血单个核细胞分离纯化。分离纯化的单核细胞经心导管在梗死相关动脉内注射治疗。干细胞移植术后进行随访1、3、6月的心电图、心脏超声测定左室射血分数(LVEF)、心肌核素显像。同时与对照组进行比较。随访结果显示：经干细胞移植治疗后的患者心功能明显改善、心电图上心肌梗死部位有小的R波、心脏内存活细胞较多。
结论：(1)脐血成为继骨髓之外的另一个MSCs来源。脐血MSCs为心肌细胞移植提供了良好的细胞供体,是心肌干细胞另一来源；(2)心肌细胞凋亡与缺氧呈时间相关性。人脐血MSCs和人的心肌细胞共同培养,能有效抵抗缺氧引起的心肌细胞凋亡；(3)通过我们的实验建立了从细胞水平评价心肌细胞凋亡的方法；(4)脐血MSCs在体外进行长期培养具有正常MSCs细胞的生长速度；(5)在体外进行长期培养扩增的脐血MSCs和经过5-AZA向心肌细胞诱导后的脐血MSCs没有发生恶性转化；(6)干细胞移植治疗后的患者心功能明显改善、心电图上心肌梗死部位有小的R波、心脏内存活细胞较多。
Objectives
1. Umbilical cord blood (UCB) as a source of hematopoietic stem cells has been successfully used in clinic for over 10 years. Could MSCs from UCB be isolated, cultured, amplified, and then induced into cardiomyocyte-like cells in vitro successfully? Using amplification and purification methods in vitro, we isolated and identified MSCs from UCB, and we investigated their biological properties after exposure to 5-azacytidine (5-AZA), a revulsant of promoting cardiomyocyte-like cell formation.2. We investigated whether cardiomyocytes resist to apoptosis caused by hypoxia after co-cultured with MSCs from UCB in vitro.3. MSCs from UCB were long-term cultured and amplified in vitro, their biological safety was observed after 5-AZA treatment.4. Granulocyte colony-stimulating factor (G-CSF) was used to mobilize bone marrow, after then, the mononuclear cells (MNCs) was isolated from peripheral blood to inject into the infarct site through cardiac catheterization. After treatment, the related indexes in those patients with acute MI were observed. Methods and Results
Methods and results
The first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and
The first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and passaged. Experiments were performed with cells from passage 3. Flow cytometry (FCM) was used to determine surface markers of cells (CD34, CD44, and CD90). After 10μmmol/L of 5-AZA treatment for 4 weeks, cardiomyocyte-like change was observed under inverted microscope. Transmission electron microscope (TEM) showed oval-shaped nucleus and bundles of filaments in the cytoplasm. Spindle dense bodies, abundant glycogen granules, large number of mitochondria and endoplasmic reticulum were found inside filaments. Cells with positive expression of GATA4 and cTnI were detected by immunofluorescence staining. Moreover, gene expression ofβ-myosin heavy chain (P-MyHC) was detected. After 5-AZZ treatment, cell growth cycle analysis and growth curve trace were performed.
Results:The surface markers of MSCs from UCB after purification and amplification in vitro were in consistent with those of bone marrow counterpart. Cells had high purity from the 3rd generation. UCB-derived MSCs could differentiate into cardiomyocytes after 5-AZZ treatment in vitro, retaining their original characteristic with the inductive dose.
The second part of the experiment:Human cardiac myocyte (HCM) ischemic injury model was established by exposure HCMs to hypoxia (95% N2-5% CO2). The results showed that hypoxia induced apoptosis of HCM in a time-dependent manner. Co-cultured with HCM in hypoxic conditions, UCB-derived MSCs could inhibit hypoxia-induced apoptosis.
The third part of the experiment:Whether malignant transformation of UCB-derived MSCs occurred or not was determined by long-term cultivation and amplification in vitro, growth curve and growth cycle analysis after 5-AZA induction for 28 d, telomerase activity, G-banding patterns of chromosomal karyotpes, tumor formation, cell cycle-related gene and tumorigenicity. No abnormal chromosomal karyotpes were observed in UCB-derived MSCs after 5-AZA induction compared with that of un-induced MSCs.
The fourth part of the experiment:Six patients with acute MI were selected in this study. One week before transplantation, G-CSF, a kind of stem cell mobilization reagent, was applied to mobilize bone marrow stem cells. MNCs were isolated and purified by cell isolation system, then were injected into the infarct site through cardiac catheterization. After stem cell transplantation, these patients were followed up for 1,3 or 6 months by ECG, left ventricular ejection fraction (LVEF) via cardiac ultrasound, and myocardial perfusion imaging (MPI). Compared with the control group, cardiac function in patients after stem cell transplantation improved significantly, and small R-waves appeared in infarct site determined by ECG, with more survival cells.
Conclusions
1. Umbilical cord blood is another source of MSCs other than bone marrow. UCB-derived MSCs provide a good cell donor for cardiomyocyte transplantation, which is another source of cardiac stem cells.
2. Apoptosis of cardiomyocytes and hypoxia was time-related. Co-culture of human UCB-derived MSCs and HCM can resist against hypoxia-induced myocardial apoptosis effectively.
3. The method of evaluating cardiomyocyte apoptosis at cellular level is established.
4. UCB-derived MSCs in vitro have the similar growth rate with normal MSCs.
5. No malignant transformation occurs in UCB-derived MSCs before and after 5-AZA treatment.
6. The stem cell transplantation improves patients' cardiac function significantly, and small R-waves are in infarct site on ECG, with more survival cells.
方法与结果
第一部分：提取健康妊娠产妇分娩的胎儿脐带血,分离单个核细胞进行培养、传代,取第3代以后细胞,用流式细胞仪直接标记分析CD34、CD44、CD90细胞表面标志。用10μmmol/L的5-AZA诱导4周后,倒置显微镜下观察细胞呈类心肌细胞样改变、透视电镜观察：细胞核呈椭圆形,胞浆内可见成束的肌丝,肌丝内可见梭形密体、丰富的糖原颗粒、大量的线粒体、内质网发达。免疫荧光组化染色体可见：GATA4, cTnI表达阳性的细胞。并检测细胞β-肌球蛋白重链(β-MyHC)基因的表达。5-AZZ作用后细胞进行生长周期分析以及生长曲线描记。结果显示体外纯化扩增后的脐血间充质干细胞含有与骨髓来源的间充质干细胞相同的细胞表面标志,第三代以后细胞纯度较高,经5-AZA在体外诱导培养,可形成心肌样细胞,在诱导剂量作用后,基本的生物学特性未发生改变。
第二部分：将人的心肌细胞置于95%N2-5%CO2缺氧环境下培养造成模拟心肌缺血损伤模型。实验结果显示细胞凋亡与缺氧呈时间依赖性,将人的心肌细胞和人脐血MSCs在缺氧环境下共同培养,脐血MSCs具有抵抗缺氧引起的心肌细胞凋亡的能力。
第三部分：通过对体外长期培养、扩增的脐血MSCs细胞和经5-AZA诱导作用28天后的MSCs细胞的生长曲线和生长周期分析、细胞核型分析、端粒酶活性检测、细胞周期相关基因表达的检测以及细胞成瘤实验来评价试验用脐血MSCs是否发生恶性转化。结果证实：经离体培养、扩增脐血MSCs和经5-AZA诱导分化成心肌样细胞后不存在基因突变,不具有成瘤性。
第四部分：选择6例急性心肌梗死的病人,在移植前1周,应用干细胞动员剂,重组人粒细胞集落刺激因子(G-CSF)对进行骨髓动员,然后用细胞分离仪对外周血单个核细胞分离纯化。分离纯化的单核细胞经心导管在梗死相关动脉内注射治疗。干细胞移植术后进行随访1、3、6月的心电图、心脏超声测定左室射血分数(LVEF)、心肌核素显像。同时与对照组进行比较。随访结果显示：经干细胞移植治疗后的患者心功能明显改善、心电图上心肌梗死部位有小的R波、心脏内存活细胞较多。
结论：(1)脐血成为继骨髓之外的另一个MSCs来源。脐血MSCs为心肌细胞移植提供了良好的细胞供体,是心肌干细胞另一来源；(2)心肌细胞凋亡与缺氧呈时间相关性。人脐血MSCs和人的心肌细胞共同培养,能有效抵抗缺氧引起的心肌细胞凋亡；(3)通过我们的实验建立了从细胞水平评价心肌细胞凋亡的方法；(4)脐血MSCs在体外进行长期培养具有正常MSCs细胞的生长速度；(5)在体外进行长期培养扩增的脐血MSCs和经过5-AZA向心肌细胞诱导后的脐血MSCs没有发生恶性转化；(6)干细胞移植治疗后的患者心功能明显改善、心电图上心肌梗死部位有小的R波、心脏内存活细胞较多。
Objectives
1. Umbilical cord blood (UCB) as a source of hematopoietic stem cells has been successfully used in clinic for over 10 years. Could MSCs from UCB be isolated, cultured, amplified, and then induced into cardiomyocyte-like cells in vitro successfully? Using amplification and purification methods in vitro, we isolated and identified MSCs from UCB, and we investigated their biological properties after exposure to 5-azacytidine (5-AZA), a revulsant of promoting cardiomyocyte-like cell formation.2. We investigated whether cardiomyocytes resist to apoptosis caused by hypoxia after co-cultured with MSCs from UCB in vitro.3. MSCs from UCB were long-term cultured and amplified in vitro, their biological safety was observed after 5-AZA treatment.4. Granulocyte colony-stimulating factor (G-CSF) was used to mobilize bone marrow, after then, the mononuclear cells (MNCs) was isolated from peripheral blood to inject into the infarct site through cardiac catheterization. After treatment, the related indexes in those patients with acute MI were observed. Methods and Results
Methods and results
The first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and
The first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and passaged. Experiments were performed with cells from passage 3. Flow cytometry (FCM) was used to determine surface markers of cells (CD34, CD44, and CD90). After 10μmmol/L of 5-AZA treatment for 4 weeks, cardiomyocyte-like change was observed under inverted microscope. Transmission electron microscope (TEM) showed oval-shaped nucleus and bundles of filaments in the cytoplasm. Spindle dense bodies, abundant glycogen granules, large number of mitochondria and endoplasmic reticulum were found inside filaments. Cells with positive expression of GATA4 and cTnI were detected by immunofluorescence staining. Moreover, gene expression ofβ-myosin heavy chain (P-MyHC) was detected. After 5-AZZ treatment, cell growth cycle analysis and growth curve trace were performed.
Results:The surface markers of MSCs from UCB after purification and amplification in vitro were in consistent with those of bone marrow counterpart. Cells had high purity from the 3rd generation. UCB-derived MSCs could differentiate into cardiomyocytes after 5-AZZ treatment in vitro, retaining their original characteristic with the inductive dose.
The second part of the experiment:Human cardiac myocyte (HCM) ischemic injury model was established by exposure HCMs to hypoxia (95% N2-5% CO2). The results showed that hypoxia induced apoptosis of HCM in a time-dependent manner. Co-cultured with HCM in hypoxic conditions, UCB-derived MSCs could inhibit hypoxia-induced apoptosis.
The third part of the experiment:Whether malignant transformation of UCB-derived MSCs occurred or not was determined by long-term cultivation and amplification in vitro, growth curve and growth cycle analysis after 5-AZA induction for 28 d, telomerase activity, G-banding patterns of chromosomal karyotpes, tumor formation, cell cycle-related gene and tumorigenicity. No abnormal chromosomal karyotpes were observed in UCB-derived MSCs after 5-AZA induction compared with that of un-induced MSCs.
The fourth part of the experiment:Six patients with acute MI were selected in this study. One week before transplantation, G-CSF, a kind of stem cell mobilization reagent, was applied to mobilize bone marrow stem cells. MNCs were isolated and purified by cell isolation system, then were injected into the infarct site through cardiac catheterization. After stem cell transplantation, these patients were followed up for 1,3 or 6 months by ECG, left ventricular ejection fraction (LVEF) via cardiac ultrasound, and myocardial perfusion imaging (MPI). Compared with the control group, cardiac function in patients after stem cell transplantation improved significantly, and small R-waves appeared in infarct site determined by ECG, with more survival cells.
Conclusions
1. Umbilical cord blood is another source of MSCs other than bone marrow. UCB-derived MSCs provide a good cell donor for cardiomyocyte transplantation, which is another source of cardiac stem cells.
2. Apoptosis of cardiomyocytes and hypoxia was time-related. Co-culture of human UCB-derived MSCs and HCM can resist against hypoxia-induced myocardial apoptosis effectively.
3. The method of evaluating cardiomyocyte apoptosis at cellular level is established.
4. UCB-derived MSCs in vitro have the similar growth rate with normal MSCs.
5. No malignant transformation occurs in UCB-derived MSCs before and after 5-AZA treatment.
6. The stem cell transplantation improves patients' cardiac function significantly, and small R-waves are in infarct site on ECG, with more survival cells.
引文
1 Alemida-Porada G, PoradaC, Zanjani ED. Adult stemm cell plasticity and methods of detection[J]. Rev Clin Exp Hematol,2001,5(1):26.
2 Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization [J].Circ Res, 1999,85:221-228.
3 Bittira B, Kuang J Q, AbdulazizAK, Al-Khaldi A, Shum-Tim D, Chiu RC, In vitro preprogramming of marrow stromal cells for myocardialregeneration. Ann. Thorac. Surg,2002,74(4):1154-1 160.
4 Blackburn, E. H. (2001). Switching and signaling at the telomere, Cell 106,661-663.
5 Bowen FW, Hattori T, Narula N, et al. Reappearance of myocytes in ovine infarcts produced by six hours of complete ischemia followed by reperfusion. Am Thorac Surg,2001,71:1845-1855.
6 Chadeneau et al.,1995; Chadeneau, C., Ha, K., Hirte, H. W., Gallinger, S.,and Bacchetti,S. (1995). Telomerase activity associatd wth acquisi-tion of malignancy in human colorectal cancer. Cancer Res.55, 2533-2536.
7 Colucci W S. Apoptosis in t he heart [J]. New Engl J Med,1996,335: 1224-1226.
8 Colucci WS. Molecular and cellular mechanisms of myocardial failure. Am J Cardiol,1997,80(11A):15L-25L.
9 Conget PA, Minguell JJ. J Cell Physiol,1999,181:67-73.
10 Dahse, R., Fiedler, W., and Ernst, G. (1977). Telomeres and telomerase: biological and clinical importance. Clin. Chem.43,708-714.
11 Deten A, Holzl A, Leicht M, et al. Changes in extracellular matrix and in transforming growth factor beta isofrms after coronary artery ligation in rats. J Moll Cell Cardiol,2001,33:1191-1207.
12 Dlppolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-relate osteogenic potential of mesenchymal stromal cells from human vertebral bone marrow. J Bone Miner Res.1999:48:913-927.
13 Ducharme A, Frantz S, Aikawa M, et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J clin Invest,2000,106:55-62.
14 Eerices A, Conget P,Minguell J. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol.2000;109:235-242.
15 Findley CM, Cudmore MJ, Ahmed A, et al. VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling. Arterioscler Thromb Vasc Biol 2007;27(12):2619-2626.
16 Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Cire Res 1996;79:949-956
17 Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovascular Research,2002,53(1):31-47.
18 Fukuda, K. Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells. Ongenit. Anom. Kyoto,2002, 42(1):1-9.
19 Fukuda, K. Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells. Ongenit. Anom. Kyoto,2002, 42(1):1-9.
20 Goodwin H S, Bicknese A R, Chien S N, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant,2001,7:2581-2588.
21 Guilherme VS, Silvio L, Joao ARA. et al. Mesenchymal stem cells diferentiateimoan endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model Circulation.2005,111:150-156.
22 Hahn,W. C., Counter, C.M., Lundberg, A. S., Beijersbergen, R. L., Brooks, M. W., and Weinberg, R.A. (1999). Creation of human tumor cells with defined genetic elements, Nature 400,464-468.
23 Hans Reinecke, Charles E Murry. Transdifferentiation of skeletal muscle stem cdls after grafting into the heart [J]. Circulation,2000, 102(18):3155.
24 Han D K M, Chaudhary P M,WrightM E, et al. MRIT,a novel deat h ef2 fector domain-containing protein, in2 teract s with caspases and BclXL and initiated cell deat h [J]. Proc Natl Acad Sci SA,1997,94:11333-11338.
25 Harris, S. L., and Levine, A.J. (2005). The p53 pathway:positive and Negative feedback loops. Oncogene 24,2899-2908.
26 Hayashida W, Horiuchi M, Zhang L, Dzau VJ. Expression of p53 and Bax genes are induce in the ischemia-reperfused rat ventricle:potential roles in myocardial apoptosis. Circulation 1996;94(suppl I):1-225
27 Henriksson and Luscher,1996 Henriksson, M., and Luscher, B. (1996). Proteins of the Myc network:essential regulators of cell growth and differentiation, Adv. Cancer Res.68,109-182.
28 Hermeking H. The MYC oncogene as a cancer drug target[J]. Curr Cancer Drug Targets,2003,3(3):163 175.
29 Hou LL, Cao H, Wang DM, et al. Induction of umbilical cord blood mesenchymal stem cells into neuron-like cells in vitro. Int J Hematol. 2003,78:256-261.
30 Huang Jing-ling, Yang Shui-xiang. Ddifferentiation of mesenchymal stem cells derived from human umbilical cord into cardiomyocytes. [J].中国组织工程研究与临床康复,2008：12(47)：9367-9370.
31 Huss R, Lange C, Weissinger EM, Kolb HJ, Thalmeier K. Evidence of peripheral blood-derived, plastic-adherent CD34/low hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem Cells,2000,18:252-260.
32 Jackson KA, Majka SM, Wang H, et al. J Clin Invest,2001,107(11): 1395-1402.
33 James TN. Normal and abnormal consequences of apoptosis in the human heart. From postnatal morphogenesis to paroxysmal arrhythmias. Circulation 1994;90:556-573
34 Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bonemarrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines [J]. Circulation,2001,1046-1052.
35 Kaplan RC, Strickler HD, Rohan TE, et al. Insulin-like growth factors and coronary heart diseasee·Cardiol Rev,2005,13(1):35-39.
36 Kathyjo A. Jackson, Susan M. Hongyu Wang, J Clin Invest. J et al. June 2001, Volume 107, Number 11,1395-1402
37 Kerr J R, Wyllie A H,Currie A R, Apoptosis:a basic biological p he2 nomenon with wide-ranging implica2 tions in tissue kinetics [J]. Br J Cancer,1972,26 (3):239-257.
38 Kim et al,2004 Kim, S. I., Park, C. S., Lee, M. S., Kwon, M. S., Jho, E. H., and Song, W. K. (2004). Cyclin-dependent kinase 2 regulates the interaction of Axin with beta-catenin, Biochem. Biophys. Res. Commun. 317,478-483.
39 Kim et al.,1994 Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C.B., Wet, M.D., Ho, P.L.Coviello, G.M., Wright, W. E., Weinrich, S. L., and Say, J. W. (1994). Secific association of human telomerase activity with immortal cells ad cancer. Science 266,2011-2015.
40 Kim HE, Dalal SS, Young E, et al. Disruption of the myocardial extracellular matrix leads to cardiac dysfunction. J Clin Invest,2000,106:857-866.
41 Kinnaird T, Stabile E, Bumett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrinemechanisms[J].Circ Res,2004,94 (5):678-685.
42 Konieczny SF, Emersin CP.5-azacytidine induction of stable mesodermal stem cell lineages from IOTI/2 cells:evidence for regulatory genes controlling determination. Cell,1984,38:791-800.
43 Laflamme MA,Myerson D,Saffitz JE, et al.2002
44 Laflamme MA, Myerson D, Saffitz JE, et al. Evidence for cardiomyocyte reppulation by extracardiac progenitors in transplanted human hearts [J].Circ Res,2002,90;634-640.
45 Laustsen PG, Russell SJ, Cui L, et al. Essential roal of insulin and insulin-like growth factor21 receptor signaling in cardiac development and function[J]. Mol Cell Biol,2007,27 (5):1649 1664.
46 Leor J, Patterson M, Quinones M, et al. Circulation,1996; 94(9 Suppl):Ⅱ332-Ⅱ336.
47Lockshin RA. Programmed cell death:history and future of a concept [J]. Soc Biol,2005,199 (3):169-173.
48 Li PK, Mickle DA, Weisel RD, et al. Circulation,1997; 96(9 Suppl): Ⅱ179-Ⅱ186
49 Li RK, Mickle DA, Weisel RD, Rao V, Jia ZQ. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg,2001,72(6):1957-1963.
50 Li RK, Jia ZQ, Weisel RD, et al. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol, 1999,31:513-522.
51 Li RK, Jia ZQ, Weisel RD, et al. Ann Thorac Surg,1996; 62(3):654-661
52 Li RK, Mickle DA, Weisel RD, et al, Circ Res,1996; 78 (2):283-288
53 Liu D, Si H, Reynolds KA, et al. Dehydro epiandro sterone vascular endothelial cells against apoptosis Protects through a Galphai protein-dependent activation of phosphatidylinositol 3-kinase/Akt and regulation of antiapoptotic Bcl-2 expression. Endocrinology 2007;148(7):3068-3076.
54 Maclellan, W. R, and Schneider, M. D.2000. Genetic dissection of cardiac growth control pathways. Annu. Rev. Physiol.62:289-319.
55 Maclellan, W. R, and Schneider, M. D.2000. Genetic dissection of cardiac growth control pathways. J. Annu. Rev. Physiol.62:289-319.
56 Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest,1999,103(5):697-705.
57 Mark F. Pittenger and Bradley. Martin. Mesenchymal Stem Cells and Their Potential as Cardiac Therapeutics. J. Circ. Res,2004,95:9-20
58 Maruyama K, Mori Y, Murasawa S, et al. Interleukin-1 β upregulates cardiac expression of vascular endothelial growth factor and its receptor KDR/flk-1 via activation of protein tyrosine kinases [J]. J Mol cell Cardiol,1999,31;607-617.
59 Massiliano G, Huamei H, Olin D L, et al. Paracfine action accounts for marked protection of ischemie heart by Akt-modified mesenchymal stem cells. Nat Med,2005,11:367-368.
60 Menasche P, Hagege AA, Scorsin M, et al. Myoblast transplantation for heart failure. Lancet,2001,357(9252):279-280.
61 Misao J, Hayakawa Y, Ohno M, et al. Expression of bcl-2 protein, an inhibitor of apoptosis and Bax, an accelerator of apoptosis, in ventricular myocytes of hhuman hearts with myocardial infarction. Circulation 1996;94:1506-1512
62 Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardic hypertrophy. Cell.1998:93:215-228.
63 Morin, G. B. (1989). The human telomere terminal transferase enzyme is a nibonuleoprotein that synthesizes TTAGGG repeats. Cell 59,521-529.
64 Mu and Wei,2002 Mu, J., and Wei, L. X. (2002). Telomere and telomerase in oncology. Cell Res.12,1-7.
65 O'Kane, Ferguson MW. Trasforming factor beta-s and wound healing. In J Biocem Cell Biol,1997,29:63-78.
66 Olivetti G, Melissari M, Capasso JM, Anversa P. Cardiomyopathy of the aging human heart. Myocyte loss and cellular hypertrophy. Cir Res 1991;68:1560-1568)
67 Orlic D, Kajstura J, Chimenti S, et al. bone marrow cells regenerate infarcted myocardium [J]. Nature,2001,410:701-705.
68 Orlic D, Kajstura J, Chimenti S, et al. mobilized bone marrowcellsrepair the infracted heart, improving function and survival [J]. proc Natl Acad Sci USA,2001,98:10344-10349.
69 Orlic D, Kajstura J, Chimenti S, et al. Proc Natl Acad Sci USA,2001, 98(18):10344-10349.
70 Oscar K. Lee, Tom K. Kuo, Wei-Ming Chen, Kuan-Der Lee, Shie-Liang Hsieh, and Tain-Hsiung Chen. Isolation of multipotent Mesenchymal stem cells from umbilical cord blood. Blood.2004:103:1669-1675.
71 Pelengaris S, Khan M. The many faces of c myc [J]. Archives of Biochemistry and Biophysics,2003,416 (2):129 137.
72 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science,1999,284:143-147.
73 Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart [J].N Engl J Med,2002,346;5-10.
74 Rangarajan, A., and Weiberg, R. A. (2003). Opinion:comparative biology of mouse versus human cells:modling human cancer in mice. Nat. Rev. Cancer 3,952-959.
75 Raphael J, Abedat S, Rivo J, et al. Volatile anesthetic preconditioning attenuates myocardial apoptosis in rabbits after regional ischemia and reperfusion via Akt signaling and modulation of Bcl-2 family proteins. J Pharmacol Exp Ther 2006;318(1):186-194.
76 Rehman J, Traktuev D, Li J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004;109(10):1292-8.
77 Ren-Ke Li, Richard D Weisel, Donald A B Mickle et al. Autologous porcine heart cell trantnsplantation improved heart function after a myocardial infarction [J]. The Journal of Thoracic and Cardiovascular Surgery,2000,119(1); 62-67.
78 Robertson KD, Jones PA. Methylation past and present. Carcinogenesis, 2000,21(3):461-467.
79 Rosenberg et al,1995; Rosenberg, A. R., Zindy, F., Deis, F. L., Mouly, H., Metezeau, P., Brechot, C., and Lamas, E. (1995). Overexpression of human cyclin A advances entry into S phase. Oncogene 10,1501-1509.
80 Ross RS, Borg TK. Integrins and myocardium. Circ Res,2001,88:1112-1119.
81 Ruggero D M, Monica B, Maria G C, et al. Functional expression of Fas and Fas ligand on human gut lamina propria T lymp hocytes:A potential role for t he acidic sp hingomyelinase pat hway in normal immunoregulation[J].J Clin In vest,1996,97 (2):316-322.
82 Sakai T, Li RK, Weisel RD, et al. Autologous heart cell transplantation improves cardiac function after myocardial injyry[J]. Ann Thorac Surg,1999,68:2074-2081.
83 Schwartz S M. Cell deat h and t he ca2 soase cascade [J] Circulation,1998,97:227-229.
84 Schwetz I, Naliboff B, Munakata J, et al. Anti-hyperalgesic effect ofoctreotide in patientswith irritable bowel syndrome Aliment Pharmacol Ther,2004,19 (1):23-131.
85 Scorsin M, Marotte F, Sabxi A, et al. Circulation,1996; 94(Suppl 11) Ⅱ337-Ⅱ340
86 Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res,1999,250:273-283.
87 Shake JG, Gruber PJ, Baumqartner WA, et al. Mesenchymal stem cells implantation in a swin myocardial infarctmodel:engraftment and functional effects[J]. Ann Thorac Surg,2002,73 (6):1919-1925.
88 Shay, J.W., and Wright, W.E. (2001). When do telomeres matter. Sience 291,839-840.
89 Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells [J]. Blood,1998,92:362-367.
90 Shintani S, Murohara T, Ikeda H, et al. Circulation,2001,9.
91 Soinale FG, Coker ML, Bond BR, et al. Myocardial matrix degradation and metalloproteinase activation in the failing heart:a potential therapeutic target. Cardiovase Res,2000,46:225-238.
92 Soonpaa MH, Koh GY, Klug MG, rt al. Formation of nascent intercalated disks between graftedfetal cardiomocytes and host myocardium[J]. Science,1994,264:98-101.
93 Stamm C, Westphal B, Kleine HD, et al. Autologous Bone-marrow stem2cell transp lantation for myocardial regeneration. [J]. Lancet,2003, 361 (9351):45-46.
94 Steele, R.J., and Lane, D. P. (2005). P53 in cancer:a paradigm for modern management of cancer. Surgeon 3,197-205.
95 Sutton MG, Sharpo N. Left ventricular remodeling after myocardial Infarction:pathophysiology an d therapy. Circulation,2000, 101:2981-2988.
96 Thompson NL, Bazoberry F, Speir EH, et al. Transforming growth factor beta-1 in acute myocardial infarctiong in rats. Growth Factor, 1988,1:91-99.
97 Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cells lines derived from human blastocysts[J]. Seience,1998, 282(5391):1145-1147.
98 Toma C, Pittenger MF, Cahill KS, et al. human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart [J]. Circulation,2002,105;93-98.
99 Toma, C., M. F. Pittenger, K. S. Cahill, B.J.Byrne, P.D.Kessler, Human mesenchymal stem cells differentiate to a cardiornyocyte phenotype in the adult murine heart. Circulation,2002,:105(1):93-98.
100 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-Ⅱ256.
101 Tomita, S., D.A. Mickle, R. D. Weisel, Z.Q. Jia, L.C. Tumiati, Y. Allidina, P. Liu & R.K.Li, Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stomal cell transplantation. J Thorac Cardiovasc Surg,2002,123(6):1132-1140.
102 Tomita, S, D.A. Mickle, R.D. Weisel, Z.Q. Jia, L.C. Tumiati, Y. Allidina, P. Liu&R. K. Li. Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stromal cell transplantation. J Thorac Cardiovasc Surg,2002,123(6):1132-1140.
103 Tomita, S., D.A. Mickle, R. D. Weisel, Z.Q. Jia, L.C. Tumiati, Y. Allidina, P.Liu & R. K, Li, Improved heart function with myogenesis and angiogenesis after autologous porcine boe marrow stromal cell transplantation. J Thorac Cardiovase Surg,验证 2002,123(6):1132-1140.
104 Wadman M. Bills threaten total US ban on human cloning[J]. Nature, 2001,411(6833):3.
105 Wakitani S, Satio T, Capoan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. MuscleNerve.1995,18:1417-1426.
106 Waller EK, Olweus J. Blood,1995,85:2422.
107 Yutaka Sakakibara, Massaya Yamamoto, Kazunobu Nishi-mura, et al. Prevasculrization using cardiomyocytes transplantation in ats with ischemic cardiomyopathy[J]. Circulation,2000,102 (18):3152.
108党双锁,李林瑞,刘效恭,等.脂质体介导c-myc基因反义寡核苷酸对人肝癌细胞的生物学影响[J].中华实验外科杂志,2000,17(6)：533 534.
109涂向东,张宝珍,朱忠勇.用吐温20改进骨髓细胞染色体制备方法[J].临床检验杂志,2002,20(6)：353-379.
110《中华人民共和国药典》(2005年第五版),国家药典委员会编.
111中华人民共和国国家标准GB/T16886.3-1997《医疗器械生物学评价,第3部分,遗传毒性、致癌性和生殖毒性试验》
112中华人民共和国科学技术部.关于善待实验动物的指导性意见.
113周恒,李宇琳,孙剑,等.人骨髓基质干细胞成骨诱导后端粒酶活性的检测分析[J].组织工程与重建外科,2007,3(1)：33-35.
114朱宏生,连峰,朱伟,钟站,郑林,朱平.骨髓间充质干细胞体外转化为心肌细胞的实验研究.上海第二医科大学学报,2002,22(5)：391-395.
2 Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization [J].Circ Res, 1999,85:221-228.
3 Bittira B, Kuang J Q, AbdulazizAK, Al-Khaldi A, Shum-Tim D, Chiu RC, In vitro preprogramming of marrow stromal cells for myocardialregeneration. Ann. Thorac. Surg,2002,74(4):1154-1 160.
4 Blackburn, E. H. (2001). Switching and signaling at the telomere, Cell 106,661-663.
5 Bowen FW, Hattori T, Narula N, et al. Reappearance of myocytes in ovine infarcts produced by six hours of complete ischemia followed by reperfusion. Am Thorac Surg,2001,71:1845-1855.
6 Chadeneau et al.,1995; Chadeneau, C., Ha, K., Hirte, H. W., Gallinger, S.,and Bacchetti,S. (1995). Telomerase activity associatd wth acquisi-tion of malignancy in human colorectal cancer. Cancer Res.55, 2533-2536.
7 Colucci W S. Apoptosis in t he heart [J]. New Engl J Med,1996,335: 1224-1226.
8 Colucci WS. Molecular and cellular mechanisms of myocardial failure. Am J Cardiol,1997,80(11A):15L-25L.
9 Conget PA, Minguell JJ. J Cell Physiol,1999,181:67-73.
10 Dahse, R., Fiedler, W., and Ernst, G. (1977). Telomeres and telomerase: biological and clinical importance. Clin. Chem.43,708-714.
11 Deten A, Holzl A, Leicht M, et al. Changes in extracellular matrix and in transforming growth factor beta isofrms after coronary artery ligation in rats. J Moll Cell Cardiol,2001,33:1191-1207.
12 Dlppolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-relate osteogenic potential of mesenchymal stromal cells from human vertebral bone marrow. J Bone Miner Res.1999:48:913-927.
13 Ducharme A, Frantz S, Aikawa M, et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J clin Invest,2000,106:55-62.
14 Eerices A, Conget P,Minguell J. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol.2000;109:235-242.
15 Findley CM, Cudmore MJ, Ahmed A, et al. VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling. Arterioscler Thromb Vasc Biol 2007;27(12):2619-2626.
16 Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Cire Res 1996;79:949-956
17 Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovascular Research,2002,53(1):31-47.
18 Fukuda, K. Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells. Ongenit. Anom. Kyoto,2002, 42(1):1-9.
19 Fukuda, K. Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells. Ongenit. Anom. Kyoto,2002, 42(1):1-9.
20 Goodwin H S, Bicknese A R, Chien S N, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant,2001,7:2581-2588.
21 Guilherme VS, Silvio L, Joao ARA. et al. Mesenchymal stem cells diferentiateimoan endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model Circulation.2005,111:150-156.
22 Hahn,W. C., Counter, C.M., Lundberg, A. S., Beijersbergen, R. L., Brooks, M. W., and Weinberg, R.A. (1999). Creation of human tumor cells with defined genetic elements, Nature 400,464-468.
23 Hans Reinecke, Charles E Murry. Transdifferentiation of skeletal muscle stem cdls after grafting into the heart [J]. Circulation,2000, 102(18):3155.
24 Han D K M, Chaudhary P M,WrightM E, et al. MRIT,a novel deat h ef2 fector domain-containing protein, in2 teract s with caspases and BclXL and initiated cell deat h [J]. Proc Natl Acad Sci SA,1997,94:11333-11338.
25 Harris, S. L., and Levine, A.J. (2005). The p53 pathway:positive and Negative feedback loops. Oncogene 24,2899-2908.
26 Hayashida W, Horiuchi M, Zhang L, Dzau VJ. Expression of p53 and Bax genes are induce in the ischemia-reperfused rat ventricle:potential roles in myocardial apoptosis. Circulation 1996;94(suppl I):1-225
27 Henriksson and Luscher,1996 Henriksson, M., and Luscher, B. (1996). Proteins of the Myc network:essential regulators of cell growth and differentiation, Adv. Cancer Res.68,109-182.
28 Hermeking H. The MYC oncogene as a cancer drug target[J]. Curr Cancer Drug Targets,2003,3(3):163 175.
29 Hou LL, Cao H, Wang DM, et al. Induction of umbilical cord blood mesenchymal stem cells into neuron-like cells in vitro. Int J Hematol. 2003,78:256-261.
30 Huang Jing-ling, Yang Shui-xiang. Ddifferentiation of mesenchymal stem cells derived from human umbilical cord into cardiomyocytes. [J].中国组织工程研究与临床康复,2008：12(47)：9367-9370.
31 Huss R, Lange C, Weissinger EM, Kolb HJ, Thalmeier K. Evidence of peripheral blood-derived, plastic-adherent CD34/low hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem Cells,2000,18:252-260.
32 Jackson KA, Majka SM, Wang H, et al. J Clin Invest,2001,107(11): 1395-1402.
33 James TN. Normal and abnormal consequences of apoptosis in the human heart. From postnatal morphogenesis to paroxysmal arrhythmias. Circulation 1994;90:556-573
34 Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bonemarrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines [J]. Circulation,2001,1046-1052.
35 Kaplan RC, Strickler HD, Rohan TE, et al. Insulin-like growth factors and coronary heart diseasee·Cardiol Rev,2005,13(1):35-39.
36 Kathyjo A. Jackson, Susan M. Hongyu Wang, J Clin Invest. J et al. June 2001, Volume 107, Number 11,1395-1402
37 Kerr J R, Wyllie A H,Currie A R, Apoptosis:a basic biological p he2 nomenon with wide-ranging implica2 tions in tissue kinetics [J]. Br J Cancer,1972,26 (3):239-257.
38 Kim et al,2004 Kim, S. I., Park, C. S., Lee, M. S., Kwon, M. S., Jho, E. H., and Song, W. K. (2004). Cyclin-dependent kinase 2 regulates the interaction of Axin with beta-catenin, Biochem. Biophys. Res. Commun. 317,478-483.
39 Kim et al.,1994 Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C.B., Wet, M.D., Ho, P.L.Coviello, G.M., Wright, W. E., Weinrich, S. L., and Say, J. W. (1994). Secific association of human telomerase activity with immortal cells ad cancer. Science 266,2011-2015.
40 Kim HE, Dalal SS, Young E, et al. Disruption of the myocardial extracellular matrix leads to cardiac dysfunction. J Clin Invest,2000,106:857-866.
41 Kinnaird T, Stabile E, Bumett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrinemechanisms[J].Circ Res,2004,94 (5):678-685.
42 Konieczny SF, Emersin CP.5-azacytidine induction of stable mesodermal stem cell lineages from IOTI/2 cells:evidence for regulatory genes controlling determination. Cell,1984,38:791-800.
43 Laflamme MA,Myerson D,Saffitz JE, et al.2002
44 Laflamme MA, Myerson D, Saffitz JE, et al. Evidence for cardiomyocyte reppulation by extracardiac progenitors in transplanted human hearts [J].Circ Res,2002,90;634-640.
45 Laustsen PG, Russell SJ, Cui L, et al. Essential roal of insulin and insulin-like growth factor21 receptor signaling in cardiac development and function[J]. Mol Cell Biol,2007,27 (5):1649 1664.
46 Leor J, Patterson M, Quinones M, et al. Circulation,1996; 94(9 Suppl):Ⅱ332-Ⅱ336.
47Lockshin RA. Programmed cell death:history and future of a concept [J]. Soc Biol,2005,199 (3):169-173.
48 Li PK, Mickle DA, Weisel RD, et al. Circulation,1997; 96(9 Suppl): Ⅱ179-Ⅱ186
49 Li RK, Mickle DA, Weisel RD, Rao V, Jia ZQ. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg,2001,72(6):1957-1963.
50 Li RK, Jia ZQ, Weisel RD, et al. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol, 1999,31:513-522.
51 Li RK, Jia ZQ, Weisel RD, et al. Ann Thorac Surg,1996; 62(3):654-661
52 Li RK, Mickle DA, Weisel RD, et al, Circ Res,1996; 78 (2):283-288
53 Liu D, Si H, Reynolds KA, et al. Dehydro epiandro sterone vascular endothelial cells against apoptosis Protects through a Galphai protein-dependent activation of phosphatidylinositol 3-kinase/Akt and regulation of antiapoptotic Bcl-2 expression. Endocrinology 2007;148(7):3068-3076.
54 Maclellan, W. R, and Schneider, M. D.2000. Genetic dissection of cardiac growth control pathways. Annu. Rev. Physiol.62:289-319.
55 Maclellan, W. R, and Schneider, M. D.2000. Genetic dissection of cardiac growth control pathways. J. Annu. Rev. Physiol.62:289-319.
56 Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest,1999,103(5):697-705.
57 Mark F. Pittenger and Bradley. Martin. Mesenchymal Stem Cells and Their Potential as Cardiac Therapeutics. J. Circ. Res,2004,95:9-20
58 Maruyama K, Mori Y, Murasawa S, et al. Interleukin-1 β upregulates cardiac expression of vascular endothelial growth factor and its receptor KDR/flk-1 via activation of protein tyrosine kinases [J]. J Mol cell Cardiol,1999,31;607-617.
59 Massiliano G, Huamei H, Olin D L, et al. Paracfine action accounts for marked protection of ischemie heart by Akt-modified mesenchymal stem cells. Nat Med,2005,11:367-368.
60 Menasche P, Hagege AA, Scorsin M, et al. Myoblast transplantation for heart failure. Lancet,2001,357(9252):279-280.
61 Misao J, Hayakawa Y, Ohno M, et al. Expression of bcl-2 protein, an inhibitor of apoptosis and Bax, an accelerator of apoptosis, in ventricular myocytes of hhuman hearts with myocardial infarction. Circulation 1996;94:1506-1512
62 Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardic hypertrophy. Cell.1998:93:215-228.
63 Morin, G. B. (1989). The human telomere terminal transferase enzyme is a nibonuleoprotein that synthesizes TTAGGG repeats. Cell 59,521-529.
64 Mu and Wei,2002 Mu, J., and Wei, L. X. (2002). Telomere and telomerase in oncology. Cell Res.12,1-7.
65 O'Kane, Ferguson MW. Trasforming factor beta-s and wound healing. In J Biocem Cell Biol,1997,29:63-78.
66 Olivetti G, Melissari M, Capasso JM, Anversa P. Cardiomyopathy of the aging human heart. Myocyte loss and cellular hypertrophy. Cir Res 1991;68:1560-1568)
67 Orlic D, Kajstura J, Chimenti S, et al. bone marrow cells regenerate infarcted myocardium [J]. Nature,2001,410:701-705.
68 Orlic D, Kajstura J, Chimenti S, et al. mobilized bone marrowcellsrepair the infracted heart, improving function and survival [J]. proc Natl Acad Sci USA,2001,98:10344-10349.
69 Orlic D, Kajstura J, Chimenti S, et al. Proc Natl Acad Sci USA,2001, 98(18):10344-10349.
70 Oscar K. Lee, Tom K. Kuo, Wei-Ming Chen, Kuan-Der Lee, Shie-Liang Hsieh, and Tain-Hsiung Chen. Isolation of multipotent Mesenchymal stem cells from umbilical cord blood. Blood.2004:103:1669-1675.
71 Pelengaris S, Khan M. The many faces of c myc [J]. Archives of Biochemistry and Biophysics,2003,416 (2):129 137.
72 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science,1999,284:143-147.
73 Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart [J].N Engl J Med,2002,346;5-10.
74 Rangarajan, A., and Weiberg, R. A. (2003). Opinion:comparative biology of mouse versus human cells:modling human cancer in mice. Nat. Rev. Cancer 3,952-959.
75 Raphael J, Abedat S, Rivo J, et al. Volatile anesthetic preconditioning attenuates myocardial apoptosis in rabbits after regional ischemia and reperfusion via Akt signaling and modulation of Bcl-2 family proteins. J Pharmacol Exp Ther 2006;318(1):186-194.
76 Rehman J, Traktuev D, Li J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004;109(10):1292-8.
77 Ren-Ke Li, Richard D Weisel, Donald A B Mickle et al. Autologous porcine heart cell trantnsplantation improved heart function after a myocardial infarction [J]. The Journal of Thoracic and Cardiovascular Surgery,2000,119(1); 62-67.
78 Robertson KD, Jones PA. Methylation past and present. Carcinogenesis, 2000,21(3):461-467.
79 Rosenberg et al,1995; Rosenberg, A. R., Zindy, F., Deis, F. L., Mouly, H., Metezeau, P., Brechot, C., and Lamas, E. (1995). Overexpression of human cyclin A advances entry into S phase. Oncogene 10,1501-1509.
80 Ross RS, Borg TK. Integrins and myocardium. Circ Res,2001,88:1112-1119.
81 Ruggero D M, Monica B, Maria G C, et al. Functional expression of Fas and Fas ligand on human gut lamina propria T lymp hocytes:A potential role for t he acidic sp hingomyelinase pat hway in normal immunoregulation[J].J Clin In vest,1996,97 (2):316-322.
82 Sakai T, Li RK, Weisel RD, et al. Autologous heart cell transplantation improves cardiac function after myocardial injyry[J]. Ann Thorac Surg,1999,68:2074-2081.
83 Schwartz S M. Cell deat h and t he ca2 soase cascade [J] Circulation,1998,97:227-229.
84 Schwetz I, Naliboff B, Munakata J, et al. Anti-hyperalgesic effect ofoctreotide in patientswith irritable bowel syndrome Aliment Pharmacol Ther,2004,19 (1):23-131.
85 Scorsin M, Marotte F, Sabxi A, et al. Circulation,1996; 94(Suppl 11) Ⅱ337-Ⅱ340
86 Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res,1999,250:273-283.
87 Shake JG, Gruber PJ, Baumqartner WA, et al. Mesenchymal stem cells implantation in a swin myocardial infarctmodel:engraftment and functional effects[J]. Ann Thorac Surg,2002,73 (6):1919-1925.
88 Shay, J.W., and Wright, W.E. (2001). When do telomeres matter. Sience 291,839-840.
89 Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells [J]. Blood,1998,92:362-367.
90 Shintani S, Murohara T, Ikeda H, et al. Circulation,2001,9.
91 Soinale FG, Coker ML, Bond BR, et al. Myocardial matrix degradation and metalloproteinase activation in the failing heart:a potential therapeutic target. Cardiovase Res,2000,46:225-238.
92 Soonpaa MH, Koh GY, Klug MG, rt al. Formation of nascent intercalated disks between graftedfetal cardiomocytes and host myocardium[J]. Science,1994,264:98-101.
93 Stamm C, Westphal B, Kleine HD, et al. Autologous Bone-marrow stem2cell transp lantation for myocardial regeneration. [J]. Lancet,2003, 361 (9351):45-46.
94 Steele, R.J., and Lane, D. P. (2005). P53 in cancer:a paradigm for modern management of cancer. Surgeon 3,197-205.
95 Sutton MG, Sharpo N. Left ventricular remodeling after myocardial Infarction:pathophysiology an d therapy. Circulation,2000, 101:2981-2988.
96 Thompson NL, Bazoberry F, Speir EH, et al. Transforming growth factor beta-1 in acute myocardial infarctiong in rats. Growth Factor, 1988,1:91-99.
97 Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cells lines derived from human blastocysts[J]. Seience,1998, 282(5391):1145-1147.
98 Toma C, Pittenger MF, Cahill KS, et al. human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart [J]. Circulation,2002,105;93-98.
99 Toma, C., M. F. Pittenger, K. S. Cahill, B.J.Byrne, P.D.Kessler, Human mesenchymal stem cells differentiate to a cardiornyocyte phenotype in the adult murine heart. Circulation,2002,:105(1):93-98.
100 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-Ⅱ256.
101 Tomita, S., D.A. Mickle, R. D. Weisel, Z.Q. Jia, L.C. Tumiati, Y. Allidina, P. Liu & R.K.Li, Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stomal cell transplantation. J Thorac Cardiovasc Surg,2002,123(6):1132-1140.
102 Tomita, S, D.A. Mickle, R.D. Weisel, Z.Q. Jia, L.C. Tumiati, Y. Allidina, P. Liu&R. K. Li. Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stromal cell transplantation. J Thorac Cardiovasc Surg,2002,123(6):1132-1140.
103 Tomita, S., D.A. Mickle, R. D. Weisel, Z.Q. Jia, L.C. Tumiati, Y. Allidina, P.Liu & R. K, Li, Improved heart function with myogenesis and angiogenesis after autologous porcine boe marrow stromal cell transplantation. J Thorac Cardiovase Surg,验证 2002,123(6):1132-1140.
104 Wadman M. Bills threaten total US ban on human cloning[J]. Nature, 2001,411(6833):3.
105 Wakitani S, Satio T, Capoan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. MuscleNerve.1995,18:1417-1426.
106 Waller EK, Olweus J. Blood,1995,85:2422.
107 Yutaka Sakakibara, Massaya Yamamoto, Kazunobu Nishi-mura, et al. Prevasculrization using cardiomyocytes transplantation in ats with ischemic cardiomyopathy[J]. Circulation,2000,102 (18):3152.
108党双锁,李林瑞,刘效恭,等.脂质体介导c-myc基因反义寡核苷酸对人肝癌细胞的生物学影响[J].中华实验外科杂志,2000,17(6)：533 534.
109涂向东,张宝珍,朱忠勇.用吐温20改进骨髓细胞染色体制备方法[J].临床检验杂志,2002,20(6)：353-379.
110《中华人民共和国药典》(2005年第五版),国家药典委员会编.
111中华人民共和国国家标准GB/T16886.3-1997《医疗器械生物学评价,第3部分,遗传毒性、致癌性和生殖毒性试验》
112中华人民共和国科学技术部.关于善待实验动物的指导性意见.
113周恒,李宇琳,孙剑,等.人骨髓基质干细胞成骨诱导后端粒酶活性的检测分析[J].组织工程与重建外科,2007,3(1)：33-35.
114朱宏生,连峰,朱伟,钟站,郑林,朱平.骨髓间充质干细胞体外转化为心肌细胞的实验研究.上海第二医科大学学报,2002,22(5)：391-395.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |