摘要
目的探讨转化生长因子β(TGF-β)对骨髓间充质干细胞(MSCs)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MSCs,应用TGF-β1对第二代MSCs定向诱导,相差显微镜观察细胞形态学变化,应用免疫细胞化学技术检测诱导后4周MSCs结蛋白(desmin),α-横纹肌肌动蛋白(α-sarcomeric actin)、心肌肌钙蛋白T(CTnT)及p38MAPK的表达情况,并根据CTnT这一心肌源性特异性标记物的阳性率,分析心肌细胞的转化率;同时,以半定量RT-PCR方法在诱导后7、21、28d检测细胞心肌早期转录因子GATA-4和心肌特异性肌凝蛋白重链α-MHC的表达。结果①加入TGF-β1诱导后的MSCs,于7d时形态已转变成类长柱形,28d时细胞体积则变小,呈条梭状排列,可观察到类肌管样结构。②TGF-β1诱导4周后的MSCs均可见desmin、α-sarcomeric actin及p38MAPK阳性细胞。诱导组的心肌样细胞转化率均显著高于对照组(P均<0.01)。③RT-PCR结果显示,诱导组细胞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。结论骨髓间充质干细胞在TGF-β诱导下可定向分化为心肌样细胞。
Objective To explore the effect of differentiation of rat bone marrow mesenchymal stem cells(MSCs) into cardiomyocyte-like cells in vitro by using transforming growth factor beta(TGF-β).Methods SD rat MSCs were isolated from rat bone marrow and cultured;then the 2nd-generation MSCs were induced by TGF-β1 for 72 h.The cultured cells were observed with phase-contrast microscope for morphological changes.The immunohistochemical technique was used to detect the expressions of desmin,α-sarcomeric actin,CTnT and p38MAPK.GATA-4 and α-MHC expressions were detected by relative quantitative RT-PCR after 7,21 and 28 days of induction,respectively.Results After induction by TGF-β1 for 28 days,MSCs showed fusiform shape,orientating with one accord and forming myotubule-like structure.MSCs induced by TGF-β1 for 28 days could be identified by the positive staining for desmin,α-sarcomeric actin and p38MAPK.The positive rates were all higher than those in the control group.The number was lower in the control group than in the treatment group(P<0.01).Relative quantitative RT-PCR showed that in the treatment group GATA-4 expression was weak after 7 days of induction,increased after 21 days of induction and decreased after 28 days of induction.By contrast,α-MHC had no expression after 7 days of induction,but showed weak and obvious expression after 21 days and 28 days of induction,respectively.Conclusion TGF-β may induce MSCs to acquire cardiogenic phenotype.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2012年第4期416-420,455,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
河北省自然科学基金资助项目(No.C071101)
河北省教育厅科学研究计划项目(No.2008102)~~
关键词
骨髓间充质干细胞
TGF-Β
心肌细胞
细胞分化
细胞培养
marrow mesenchymal stem cells(MSCs)
transforming growth factor beta(TGF-β)
cardiomyocyte
differentiation
cell culture
