成纤维细胞生长因子2和丹参酮ⅡA在大鼠骨髓间充质干细胞向心肌样细胞分化中的作用

doi:10.16098/j.issn.0529-1356.2020.04.008

摘要/Abstract

摘要：

目的探讨成纤维细胞生长因子2（FGF-2）和丹参酮ⅡA（tanshinoneⅡA）在大鼠骨髓间充质干细胞（BMSCs）向心肌样细胞分化中的作用。方法体外分离、培养BMSCs，利用流式细胞术进行鉴定。实验分组：对照组（不加任何诱导剂）、FGF-2组、丹参酮ⅡA组及两者联合诱导组。MTT检测诱导后的活性及增殖情况；Real-time PCR检测早期心肌转录因子GATA-4、Nkx2.5的表达；免疫细胞化学染色法检测缝隙连接蛋白43(Cx43)以及心肌肌钙蛋白Ⅰ（cTnI）的表达；免疫荧光化学染色法检测结蛋白(desmin)、原肌球蛋白（Tm）的表达；Western blotting检测结蛋白、Tm的表达。结果各诱导组较对照组增殖明显。与对照组相比，诱导组GATA-4和Nkx2.5基因的表达增强，联合组表达量最高，差异具有统计学意义（P＜0.05）。联合诱导组各标记物Cx43、cTnI、结蛋白、Tm的阳性表达率高于FGF-2及丹参酮ⅡA单独诱导组，差异具有统计学意义（P＜0.05）。Western blotting结果显示，联合诱导组结蛋白、Tm的表达量明显高于其他实验组，差异具有统计学意义（P＜0.05）。透射电子显微镜结果显示，诱导后细胞的细胞核居中，细胞质中可见肌丝、粗面内质网、线粒体和核糖体。结论 FGF-2和丹参酮ⅡA均能促进BMSCs增殖，诱导BMSCs分化为心肌样细胞，两者联合诱导的效果较其他组更佳。

关键词: 骨髓间充质干细胞, 心肌样细胞, 成纤维细胞生长因子2, 丹参酮 Ⅱ A, 流式细胞术, 免疫印迹法, 大鼠

Abstract:

Objective To investigate the effects of fibroblast growth factor 2 (FGF-2) combined with tanshinoneⅡA on differentiation of rat bone marrow mesenchymal stem cells (BMSCs）into cardiomyocyte-like cells. Methods BMSCs were isolated and cultured. The cultured cells were identified by flow cytometry, BMSCs were divided into experimental control group, FGF-2 group, tanshinoneⅡA group and the combined induction group. The activity and value of BMSCs were detected by MTT. The Real-time PCR method was used to detect BMSCs. Expression of early myocardial transcription factors GATA-4 and Nkx2.5; Immunocytochemical staining for detection of connexin43（Cx43）and cardiac troponin-Ⅰ(cTnI);Immunofluorescence staining for detection of desmin and Tm; The expression of desmin and tropomyosin（Tm）was detected by Western blotting. Results The cell activity and proliferation after induction were good. The expression of GATA-4 and Nkx2.5 in the induction group were stronger than that in the experimental control group, and the difference was statistically significant (P<0.05). The positive expression of Cx43 and cTnI in the induction group increased significantly. The markers of the combined group were the most obvious, and the difference was statistically significant (P<0.05). The expression of Myo-specific protein desmin and Tm in the combined induction group increased significantly, and the difference was statistically significant (P<0.05). Conclusion Both FGF-2 and tanshinoneⅡA can promote the proliferation of BMSCs and induce the differentiation of BMSCs into cardiomyocyte-like cells. The synergistic effect of the two is better than other groups.

Key words: Bone marrow mesenchymal stem cell, Cardiomyocyte, Fibroblast growth factor 2, TanshinoneⅡA, Flow cytometry, Western blotting, Rat

中图分类号:

R329.2

李娇吕洋刘洋王浩宇赵亚如王雪娇王海萍 . 成纤维细胞生长因子2和丹参酮ⅡA在大鼠骨髓间充质干细胞向心肌样细胞分化中的作用[J]. 解剖学报, 2020, 51(4): 520-527.

LI Jiao Lü Yang LIU Yang WANG Hao-yu ZHAO Ya-ru WANG Xue-jiao WANG Hai-ping. Role of fibroblast growth factor 2 and tanshinoneⅡA in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells[J]. Acta Anatomica Sinica, 2020, 51(4): 520-527.

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