解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (1 ): 49-54.doi: 10.3969/j.issn.0529-1356.2013.01.010

• 细胞和分子生物学 • 上一篇    下一篇

转化生长因子β1诱导骨髓间充质干细胞分化为心肌样细胞的作用 

吕洋1,王海萍1* ,刘博2,吴志刚1,霍艳丽1,高辰玮1    

  1. 1.河北北方学院组织学与胚胎学教研室，河北 张家口075000; 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:2012-03-16 修回日期:2012-06-10 出版日期:2013-02-06 发布日期:2013-02-06
  • 通讯作者: 王海萍 E-mail:haipingmimi@126.com
  • 基金资助:

    省自然科学基金资助项目

TGF-β1 induced bone marrow mesenchymal stem cells differentiate into cardiomyocyte -like cells

LÜ Yang1,WANG Hai-ping 1* ,LIU Bo2,WU Zhi-gang1,HUO Yan-li1,GAO Chen-wei1   

  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:2012-03-16 Revised:2012-06-10 Online:2013-02-06 Published:2013-02-06

摘要：

目的 探讨转化生长因子β1 (TGF-β1)对骨髓间充质干细胞（BMSCs）定向分化为心肌样细胞的影响。方法 取SD大鼠四肢骨骨髓，分离培养BMSCs，应用2、5、10、15 μg/L TGF-β1对第2代BMSCs定向诱导，相差显微镜观察细胞形态变化，应用免疫细胞化学技术检测诱导后4周BMSCs结蛋白（desmin）、α-横纹肌肌动蛋白（α-SCA）、心肌肌钙蛋白T（cTnT）及心肌连接蛋白43 (Cx43)的表达情况。根据cTnT心肌源性特异性标记物的阳性率分析心肌样细胞的转化率；应用激光扫描共焦显微镜技术，检测诱导后4周BMSCs α-SCA、cTnT的表达；以半定量RT-PCR方法在诱导后7、21和28d检测心肌转录调节因子（GATA4）和心肌特异性α-肌球蛋白重链（α-MHC）的表达。结果 1.不同浓度TGF-β1 诱导后的BMSCs，其形态在不同的时间段存在差异。15μg/L组和2μg/L组诱导7d的BMSCs形态变化不明显，与对照组类似，随后则逐渐转变成长柱形；5μg/L组诱导后的BMSCs 于7d时形态已转变成类长柱形，28d时细胞体积则变小，呈条索状排列；10μg/L组诱导后的BMSCs类似于5μg/L组，但细胞数量相对较少。2.不同浓度TGF-β1 诱导4周后的BMSCs均可见desmin、α-SCA及Cx43的阳性细胞，其中5μg/L组的阳性率均最强。各诱导组与对照组desmin、α-SCA及Cx43的阳性率差异均具有显著性（P <0.05）。3.各诱导组cTnT均有阳性表达。根据cTnT阳性率计算出的心肌样细胞转化率在5μg/L组最高，显著高于2μg/L组(P =0.028) 和15μg/L组(P =0.000)，但与10μg/L组之间差异不显著(P >0.05)。此外，各诱导组的心肌样细胞转化率均显著高于对照组(P <0.01)。4.激光扫描共焦显微镜技术检测结果显示，经TGF-β1诱导4周的BMSCs α-SCA和cTnT蛋白均定位于细胞质且呈共表达。5.RT-PCR结果显示，GATA-4于诱导后7d弱表达，21d表达增强，28d表达减弱；α-MHC在诱导后7d不表达，21d弱表达，28d表达明显。结论 TGF-β1可以诱导BMSCs获得心肌分化表型，最佳诱导浓度是5μg/L。

关键词: 骨髓间充质干细胞, 转化生长因子β1, 心肌细胞, 分化, 免疫组织化学, 大鼠

Abstract:

Objective To explore the effect of the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in vitro using different concentrations of TGF-β1(transforming growth factor beta1). Methods BMSCs of the SD rat were isolated and cultured from rat bone marrow, then the 2nd-generation BMSCs were induced by 2, 5, 10, 15μg/L TGF-β1 for 72 hours. The cultured cells were observed under a phase-contrast microscope. The immunohistochemical technique was used to examine the expression of desmin,α-sarcomeric actin, cTnT and Cx43. The laser scanning confocal microscope (LSCM) was used for the expression of α-sarcomeric actin and cTnT. GATA-4 and α-myosin heavy chain (α-MHC) expression were detected by relative quantitative RT-PCR after 7, 21, 28 days of induction respectively. Results 1.The morphous of BMSCs cell was different in each treatment group. BMSCs after being induced by 2, 15μg/L TGF-β1 on 7days were the same as the control group. BMSCs induced by 5μg/L TGF-β1 on 28 days showed a fusiform shape, orientating with one accord, forming myotubule-like structure. The morphous of BMSCs after being induced by 10μg/L TGF-β1 was the same as the 5μg/L group, but the number of cells was relatively small. 2. BMSCs induced by different concentrations of TGF-β1 on 28 days were identified by the positive staining for desmin, α-sarcomeric actin and Cx43. The positive rates were higher than that of the control group. 3. The rate of cTnT-positive cells in the 5μg/L treatment group was greater than that of the 15μg/L treatment group (P =0.000) and the 2μg/L treatment group(P =0.028). The number in the control group was lower than that of the treatment groups(P <0.01). 4. Under LSCM after immunoflurescence labeling, we found that bright red fluorescence in the cytoplasm was observed in the α-sarcomeric actinpositive cells, green fluorescence in the cytoplasm was in the cTnT-positive cells while the part that overlaps was yellow. 5.RT-PCR assessment showed that the differentiated cells began to express GATA-4 from day 7 to day 28 of differentiation and began to express α-MHC from day 21 to day 28 of differentiation. Conclusion TGF-β1 may induce BMSCs to acquire cardiogenic phenotype and 5μg/L is a suitable induced concentration.

Key words: Bone marrow mesenchymal stem cells, Transforming growth factor beta 1, Cardiomyocytes, Differentiation, Immunohistochemistry, Rat