解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (5): 620-627.doi: 10.16098/j.issn.0529-1356.2016.05.007

• 细胞和分子生物学 • 上一篇    下一篇

转化生长因子-β1和丹参酮ⅡA联合诱导骨髓间充质干细胞向心肌样细胞的分化

吕洋1 刘博2 王海萍1* 刘源1 孙微1 李柔1 陈晓依1   

  1. 1. 河北北方学院组织学与胚胎学教研室; 2. 河北北方学院附属第一医院病理科，河北 张家口 075000
  • 收稿日期:2016-03-23 修回日期:2016-04-19 出版日期:2016-10-06 发布日期:2016-10-06
  • 通讯作者: 王海萍 E-mail:haipingmimi@126.com
  • 基金资助:

    河北省自然基金资助项目;河北省中医药管理局科研计划项目;河北省自然基金资助项目;河北北方学院创新人才培育项目

Transforming growth factor-β1 combined with tanshinoneⅡA induces bone marrow mesenchymal stem cells differentiating into cardiomyocyte-like cells in vitro

LÜ Yang1 LIU Bo2 WANG Hai-ping 1* LIU Yuan1 SUN Wei1 LI Rou1 CHEN Xiao-yi1   

  1. 1. Department of Histology and Embryology, Hebei North University; 2. Department of Pathology, the First Affiliated Hospital of Hebei North University，Hebei Zhangjiakou 075000，China

  • Received:2016-03-23 Revised:2016-04-19 Online:2016-10-06 Published:2016-10-06
  • Contact: WANG Hai-ping E-mail:haipingmimi@126.com

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摘要：

目的 探讨转化生长因子-β1(TGF-β1)与丹参酮ⅡA(tanshinoneⅡA)联合诱导对大鼠骨髓间充质干细胞(BMSCs)定向分化为心肌样细胞的影响。方法 采用全骨髓贴壁法从10只SD大鼠四肢骨中分离、培养BMSCs，流式细胞术对培养的细胞进行鉴定。对第2代BMSCs作定向诱导，根据加入诱导剂的不同分为TGF-β1组、tanshinoneⅡA组、两者联合诱导组及实验对照组(不加任何诱导剂)。诱导72h后更换为常规培养基继续培养，相差显微镜观察培养细胞的形态学变化；各组培养4周后，应用免疫细胞化学染色法检测原肌球蛋白(TPM)、缝隙连接蛋白43(Cx43)以及心肌肌钙蛋白I(cTnI)的表达情况；实时定量PCR(Real-time PCR)法检测各组BMSCs在诱导培养的第1、2、4周心肌早期转录因子GATA-4、Nkx2.5的表达情况；透射电子显微镜观察分化细胞的超微结构变化。 结果 实验对照组细胞TPM、Cx43及cTnI均为弱阳性或阴性表达。与实验对照组相比，TGF-β1组、TanshinoneⅡA组及两者联合诱导组BMSCs以上各标记物的阳性表达均明显升高，差异均具有统计学意义(均 P<0.05)。其中，两者联合诱导组各标记物的阳性表达均显著高于TGF-β1及TanshinoneⅡA单独诱导组。Real-time PCR结果显示，在诱导第1周时TGF-β1组、TanshinoneⅡA组及两者联合诱导组GATA-4及Nkx2.5基因表达均最强，随后表达减弱至不表达。1周时，联合诱导组GATA-4 mRNA相对表达量是实验对照组的3.7倍，Nkx2.5 mRNA相对表达量是实验对照组的2.9倍，差异均具有统计学意义(均 P<0.05)。透射电子显微镜结果显示，各诱导组均可见分化的细胞呈杆状，胞核卵圆形，位于细胞中央，胞质中可见平行排列的肌丝、粗面内质网和线粒体等细胞器。 结论 TGF-β1、tanshinoneⅡA均可分别及联合诱导BMSCs获得心肌分化表型，且两者联合诱导效果优于单一诱导。

关键词: 骨髓间充质干细胞, 转化生长因子-β1, 丹参酮ⅡA, 心肌细胞, 流式细胞术, 免疫组织化学, 实时定量聚合酶链反应, 大鼠

Abstract:

Objective The present study aims to test the hypotheses that if transforming growth factor beta 1(TGF-β1) or tanshinoneⅡA or their combination enhance the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) towards the cardiomyogenic phenotype, and TGF-β1 combined with tanshinoneⅡA may achieve better effects than the alone group. Methods BMSCs were isolated from bones of limbs of 10 male SD rat and cultured. The 2nd-generation BMSCs were co-incubated with TGF-β1 or tanshinoneⅡA or their combination for 72 hours. The control group was BMSCs which cultured without any inductive substance. The morphological characteristics, surface antigens and mRNA expression of several transcription factors in each group were assessed. Results BMSCs were initially spindle-shaped with irregular processes. The cells were gradually increased after been inoculating for 24 hours and proliferated 1 week later. On week 4, BMSCs in the combined group showed fusiform shape, orientating with one accord, and were connected with adjoining cells forming myotube-like structures. The morphous of BMSCs in the alone induced group was similar to that in the combined group, but the amount of the cells was relatively low. Immunohistochemistry revealed the expressions of the cardiac-specific markers, tropomyosin, Cx43 and troponin Ⅰ, in the combined group were significantly higher than that of the other groups (P<0.05). The transcriptional expressions of the cardiomyocyte-specific genes GATA-4,Nkx2.5 were highest in 1week after induction, and they were all stable and significantly higher in the induced groups than that in the control group. Transmission electron microscope showed that the induced cells had a cardiomyocyte-like ultrastructure: the nucleus was round and located in the center of the cells, mitochondria and rough endoplasmic reticula were founded in the cytoplasm, and the paralleled myofilaments were seen in the cytoplasm. Conclusion Taken together, these results indicate that TGF-β1 or tanshinoneⅡA may induce BMSCs to acquire cardiogenic phenotype and TGF-β1 combined with tanshinoneⅡA may achieve better effects than the alone group.

Key words: Bone marrow mesenchymal stem cell, Transforming growth factor beta 1, TanshinoneⅡA, Cardiomyocyte, Flow cytometry, Immunohistochemistry, Real-time PCR, Rat