5-氮胞苷诱导心肌组织中c-kit+干细胞分化的实验研究

中国临床药理学与治疗学 ›› 2008, Vol. 13 ›› Issue (9): 996-1001.

5-氮胞苷诱导心肌组织中c-kit⁺干细胞分化的实验研究

姜宏¹, 施广飞²

¹南京中医药大学第一临床医学院, 南京210046, 江苏;
²南京大学医学院附属鼓楼医院心血管科, 南京210008, 江苏

收稿日期:2008-06-06 修回日期:2008-08-27 出版日期:2008-09-26 发布日期:2020-10-13
通讯作者: 施广飞, 男, 主任医师, 博士研究生导师, 研究方向:心肌干细胞的实验研究。Tel:025-83304616 E-mail:yuhonson@hotmail.com
作者简介:姜宏, 女, 博士研究生, 讲师, 研究方向:心肌干细胞的实验研究。Tel:025-85811629 E-mail:gingerjh@126.com
基金资助:
江苏省卫生厅科研项目(H200220);南京市卫生局科研项目(ZKX0206)

Empirical study on c-kit⁺ cardiac stem cells induced differentiation into cardiomyocytes by 5-aza

JIANG Hong¹, SHI Guang-fei²

¹First Clinical Medicine Collage, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, Jiangsu,China;
²Cardiovascular Division of Gulou Hospital Affiliated to Medicine College of Nanjing University, Nanjing 210008, Jiangsu, China

Received:2008-06-06 Revised:2008-08-27 Online:2008-09-26 Published:2020-10-13

摘要/Abstract

摘要： 目的:创建小鼠心肌干细胞(cardiac stem cells, CSC) 分离、培养的方法, 借助干细胞表面标记c-kit 纯化CSC, 并用5-氮胞苷诱导CSC 分化为心肌细胞。方法:用酶定时消化方法分离提取小鼠心脏组织中的细胞, 进行体外培养;免疫磁珠法纯化c-kit 阳性细胞, 并用流式细胞仪检测纯化后细胞表面标记c-kit、lin 和CD₃₄ 的表达;以免疫细胞化学染色法检测诱导后心肌特异性蛋白T、连接蛋白-43、心肌细胞特异性结蛋白;结合形态学变化, 分析不同诱导条件作用结果之间的差异。结果:从C57BL 小鼠心肌组织中分离消化所得的细胞中, 含有一种小而圆形、折光性强的细胞, 经传代培养的细胞增殖速度比原代细胞明显为快,存在较多的类似于干细胞增殖和形态学特点的细胞。用免疫磁珠法可以分选出高纯度的c-kit⁺CSC, 这种CSC 基本不表达CD₃₄ 和lin 。在不同诱导条件下, 此种CSC 可以分化为搏动的细胞, 并能表达心肌特异性蛋白。结论:从C57BL 小鼠心肌组织中可以获得CSC, 用免疫磁珠法可以分选出表型为c-kit⁺-CD₃₄^--lin^-高纯度的CSC, 经5-氮胞苷诱导或改变CSC 生长的微环境后, 此种细胞可以分化成心肌细胞, 且能形成" 心肌球" 结构。

关键词: 心肌干细胞, 诱导, 分化, 心肌细胞, 5-氮胞苷

Abstract: AIM: To find the method of dissociating and culturing the cardiac stem cells (CSC), purify CSC by cell surface markers of stem cell with c-kit, and induce CSC differentiation into cardiomyocytes by 5-azacitidine (5-aza) in vitro.METHODS: The heart cells were dissociated and extracted by zymo-timing digestion and cultured in vitro, the c-kit masculine cells as CSC markers were purified by immunomagnetic beads method.The expression such as c-kit, lin and CD₃₄ as CSC markers were detected after purification by flow cytometry.The cardiac specific troponin T (cTnT), connective-43 (Cx-43) and the specificity desmin of cardiomyocytes were detected by the method of immunocytochemical stain.The differences of the results in the different conditions were compared and analyzed combined with the morphology changes.RESULTS: The cells, which were dissociated and digested from C57BL mouse cardiac muscles, were cultured in vitro. There was a kind of small, round and high-photonasty cells.The speed of cell multiplication after serial subcultivation was faster than the primary cell.These cells were similar to the stem cell from their cell multiplication and the morphological feature.Highly purified ckit + CSC were separated by immunomagnetic beads method, but the CSC do not express CD₃₄ and lin generally.Under the different inducing conditions, these CSC could differentiate into pulsatory cells, and express the specific protein of cardiomyocytes.CONCLUSION: The CSC can be obtained from cardiac muscular tissues of C57BL mice, the highly purified CSC of c-kit +-CD₃₄^--lin^- can be separated by using the magnetic activated cell sorting (MACS) system. After induced by 5-aza or changed the microenvironment of CSC growth, the CSC can differentiate into cardiomyocytes and form " cardiomyocytes cluster" construction.

Key words: cardiacstemcell, induction, differentiation, cadiocyte, 5-azacitidine

中图分类号:

R965.2

姜宏, 施广飞. 5-氮胞苷诱导心肌组织中c-kit⁺干细胞分化的实验研究[J]. 中国临床药理学与治疗学, 2008, 13(9): 996-1001.

JIANG Hong, SHI Guang-fei. Empirical study on c-kit⁺ cardiac stem cells induced differentiation into cardiomyocytes by 5-aza[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(9): 996-1001.

参考文献

[1] Pouzer B, Vilquin JT, Hagege AA, et al. Intramyocardial transplantation of autologous myoblasts:can tissue processing be optimized [J].Circulation, 2000, 102 (19 Suppl 3):III 210-215.
[2] Connold AL, Frischknecht R, DimitrakosM, et al. The survival of embryonic cardiomyocytes transplanted into damaged host rat myocardium [J].J Muscle Res Cell Motil, 1997, 18(1):63-70.
[3] Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium [J].Nature, 2001, 410 (6829):701-705.
[4] Choi SC, Yoon J, Shim WJ, et al. 5-azacytidine induces cardiac differ-entiation of P19 embryonic stem cells [J]. ExpMol Med, 2004, 36(6):515-523.
[5] Jones PA, Taylor SM.Cellullar differentiation, cytidine analogs and DNA methylation [J].Cell, 1980, 20(1):85-93.
[6] Creusot F, Acs G, Chrisman JK.Inhibition of DNA methy ltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2-deoxycytidine [J].J Biol Chem, 1982, 257(4):2041-2048.
[7] Robertson KD, Jones PA.DNA methylation:past, present and future directions [J].Carcinogenesis, 2000, 21(2): 461-467.
[8] Wakitani S, Saito T, Caplan AI.Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine [J].Musele Nerve, 1995, 18(12):1417-l426.
[9] Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro [J].J Clin Invest, 1999, 103(5):697-705.
[10] Hakuno D, Fukuda K, Makino S, et al. Bone marrow derived regenerated cardiomyocytes express functional adrenergic and muscarinic receptors [J].Circulation, 2002, 105 (3):380-386.
[11] 赵丽丽, 屈长青, 张国华, 等.5-氮胞苷诱导大鼠脂肪间充质干细胞分化成肌细胞 [J].第四军医大学学报, 2007, 28(22):2037-2040.
[12] Xu C, Police S, Rao N, et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells [J].Circ Res, 2002, 91(6):501-508.
[13] 尹青, 赵昱, 赵秀军, 等.5-氮胞苷诱导体外培养的胚胎干细胞向心肌细胞分化 [J].基础医学与临床,2007, 27(5):549-552.
[14] Tomita S, Li RK,Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function [J].Circulation, 1999, 100(19 Suppl):II247-256.
[15] 吴铁军, 蔡振杰, 俞世强, 等.不同浓度5-氮胞苷对骨髓间质干细胞体外诱导分化的作用 [J].第四军医大学学报, 2003, 24(15):1373-1375.
[16] 贾敏, 曾秋棠, 卢沛奇, 等.骨髓间质干细胞体外诱导为心肌细胞的实验研究 [J].临床心血管病杂志, 2006, 22(6):354-356.
[17] Wobus AM, Guan K, Yang HT, et al. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation [J].Methods Mol, 2002, 185:127-156.
[18] Panteghini M.Present issues in the determination of troponins and other markers of cardiac damage [J].Clin Biochem, 2000, 33(3):161-166.
[19] Moscoso I, Centrno A, lopez E, et al. Differentiation " in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation [J]. Transplant Proc, 2005, 37(1):481-482.
[20] Lawsom-Smith MJ, McGeachie JK.The identificaion of myogenic cells in skeletal muscle, with emmphasis on the use of tritiated thymidine autoradiography and desmin antibodies [J].J Anat, 1998, 192(2):161-171.
[21] Capetanaki Y.Desmin cytoskeleton:a potential regulator of musc1e m1tochondrial behavior and function [J].Trends Cardiovasc Med, 2002, 12(8):339-348.
[22] Toyofuku T, YabubiM, Otsu K, et al. Intercellular calcium signaling via gap junction in connexin-43 transfected cells [J].J Bio Chem, 1998, 273(3):1519-1528.
[23] Tomit AS, Schuesslar RB, Berul CI, et al. Disparate effects of deficient expression of connexin-43 on atrial and ventricular conduction:evidence for chamber-specific molecular determinants of conduction [J].Circulation, 1998, 97 (7):686-689.