中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (10): 1861-1865.doi: 10.3969/j.issn.1673-8225.2012.10.033

• 干细胞与中医药 stem cells and traditional Chinese medicine • 上一篇    下一篇

5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌样细胞分化及黄芪甲苷的协同作用***☆

冼绍祥，杨忠奇，秦佳佳，黄习文，孙静和   

  1. 广州中医药大学第一临床医学院，广东省广州市  510405
  • 收稿日期:2011-06-07 修回日期:2011-08-08 出版日期:2012-03-04 发布日期:2012-03-04
  • 作者简介:冼绍祥☆，男，1962年生，广东省广州市人，汉族，2005年广州中医药大学毕业，博士，教授，主要从事中西医结合防治心血管疾病的基础和临床研究。
  • 基金资助:

    2008年国家自然科学基金面上项目(30873397)，课题名称“黄芪甲苷诱导MSCs向心肌细胞分化过程中相关受体的表达 ”；广东省自然基金资助项目(8151040701000006)，课题名称“黄芪甲苷诱导MSCs向心肌细胞分化过程中相关受体的表达”；中国博士后二等资助项目。

Bone marrow mesenchymal stem cells differentiation into cardiomyocyte-like cells induced by 5-azacytidine and astragaloside Ⅳ***☆

Xian Shao-xiang, Yang Zhong-qi, Qin Jia-jia, Huang Xi-wen, Sun Jing-he   

  1. First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou  510405, Guangdong Province, China
  • Received:2011-06-07 Revised:2011-08-08 Online:2012-03-04 Published:2012-03-04
  • About author:Xian Shao-xiang☆, M.D., Professor, First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China jiajia_bamboo@163.com
  • Supported by:

    the General Program of the National Natural Science Foundation of China in 2008, No. 30873397*; the Natural Science Foundation of Guangdong Province, No. 8151 040701000006*; Post-doctoral Second-grade Program*

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摘要：

背景：大多学者使用5-氮胞苷作为诱导剂诱导骨髓间充质干细胞向心肌细胞定向分化。
目的：观察联合应用黄芪甲苷及5-氮胞苷诱导骨髓间充质干细胞向心肌细胞定向分化中心肌细胞相关受体的表达。
方法：选用生长良好的第3代骨髓间充质干细胞，分为4组。Ⅰ组：仅更换L-DMEM培养液；Ⅱ组：100 mg/L黄芪甲苷+5 µmol/L 5-氮胞苷诱导24 h后，更换L-DMEM培养液。Ⅲ组：10 µmol/L 5-氮胞苷孵育24 h后，更换L-DMEM培养液；Ⅳ组：5 µmol/L 5-氮胞苷孵育24 h后，更换L-DMEM培养液。各组均每3 d换液1次，诱导30 d后对分化细胞进行鉴定。
结果与结论：①Ⅲ组、Ⅳ组及Ⅱ组诱导后细胞心肌细胞特异性蛋白Nkx2.5、cTnT及Desmin的表达均为阳性，与Ⅰ组比较，差异有非常显著性意义(P < 0.01)。Ⅱ组及Ⅲ组诱导后2周，镜下见cTnT、Desmin表达数量高于Ⅳ组(P < 0.01)。Nkx2.5在两组表达亦高于Ⅳ组，其中Ⅱ组与Ⅳ组比较，差异有极显著性意义(P < 0.01)，Ⅲ组与Ⅳ组比较，差异有显著性意义(P < 0.05)。Ⅰ组无上述蛋白的阳性表达。②诱导后2周，镜下可见Ⅱ组、Ⅲ组细胞出现心肌细胞样的节律性跳动，证明部分细胞在诱导因素的作用下，已向心肌细胞分化。结果表明用100 mg/L黄芪甲苷+5 µmol/L 5-氮胞苷联合诱导可产生与   10 µmol/L 5-氮胞苷相似的诱导效果，这可能与黄芪甲苷对细胞具有保护作用，促血管内皮细胞增殖作用，增强细胞对5-氮胞苷细胞毒性的耐受，上调心肌特异性蛋白的表达有关。

关键词: 黄芪甲苷, 心肌, 骨髓, 间充质干细胞, 心肌细胞

Abstract:

BACKGROUND: 5-azacytidine (5-Aza) has been frequently used to induce bone marrow mesenchymal stem cells (BMSCs) differentiation into cardiomyocyte.
OBJECTIVE: To observe expression of cardiomyocyte-related receptors in cardiomyogenic differentiation of rat BMSCs.
METHODS: BMSCs of passage three were assigned to four groups: group Ⅰ: L-DMEM solution alone was replaced; Ⅱ: L-DMEM solution was replaced after induction of 100 mg/L AST+5 µmol/L 5-Aza for 24 hours; group Ⅲ: L-DMEM solution was replaced after induction of 10 µmol/L 5-Aza for 24 hours; and group Ⅳ: L-DMEM solution was replaced after induction of 5 µmol/L 5-Aza for 24 hours. Culture medium was replaced every 3 days in each group. Differentiated cells were identified after 30 days of induction.
RESULTS AND CONCLUSION: Expression of cardiomyocyte specific proteins Nkx2.5, cTnT and Desmin was detected in groups Ⅲ, Ⅳ and Ⅱ after induction compared with group Ⅰ , with significant differences (P < 0.01). The amount of cTnT and Desmin expression expression was significantly higher in groups Ⅱ and Ⅲ compared with group Ⅳ (P < 0.01). The level of Nkx2.5 expression was significantly higher in groups Ⅱ (P < 0.01) and Ⅲ (P < 0.05) compared with group Ⅳ. No Nkx2.5, cTnT and Desmin espression was detected in group Ⅰ. After induction for 2 weeks, cells with spontaneous contractility were observed in groups Ⅱ and Ⅲ, indicating differentiation towards cardiomyocyte after induction. Results demonstrated that induction effects were similar between 100 mg/L AST+5 µmol/L 5-Aza and 10 µmol/L 5-Aza. This may contribute to cytoprotective effects of AST, which can promote vascular endothelial cell proliferation, enhance celss tolerance to 5-Aza-induced cytotoxicity and upregulate cardiac-specific protein expression.

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