催产素体外诱导大鼠骨髓间充质干细胞向心肌细胞分化

Oxytocin induces rat bone marrow mesenchymal stem cells differentiate into cardiomyocyte-like cells in vitro

目的探索催产素(OT)体外诱导骨髓间充质干细胞(MSCs)向心肌细胞分化的可行性。方法取SD大鼠四肢骨骨髓,分离培养MSCs,应用OT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、激光扫描共聚焦显微镜技术检测结蛋白,α-横纹肌肌动蛋白、心肌特异性肌钙蛋白T的表达;以半定量RT-PCR方法在诱导后7、21和28 d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。结果体外分离培养的细胞生长稳定,增殖较快,主要为纺锤形和成纤维细胞状。诱导后1周,细胞以梭形、柱状为主,部分细胞有分支,核卵圆形、居中,类似心肌细胞形态,排列方向渐趋一致。诱导后4周,细胞形态变长、增大,多呈杆状,紧密平行排列生长。分化后细胞结蛋白、α-横纹肌肌动蛋白和肌钙蛋白T均呈现阳性表达。激光共聚焦扫描显微镜观察:可见细胞质内结蛋白的表达呈红色,肌钙蛋白T的表达呈绿色,当两者同时被观察时可见其共表达的部位变成黄色。RT-PCR结果显示,GATA-4于诱导后7 d弱表达,21 d表达增强,28 d表达减弱。α-MHC在诱导后7 d不表达,21 d弱表达,28 d表达明显。结论骨髓间充质干细胞在OT诱导下可定向分化为心肌样细胞。

Objective To investigate the feasibility of inducing rat bone marrow mesenchymal stem cells（MSCs） differentiating into cardiomyocytes by oxytocin（OT）.Methods SD rat MSCs were isolated from rat bone marrow and cultured,then induced by OT for 72 h.The laser scanning confocal microscope（LSCM） was used to detect the expression of desmin,α-sarcomeric actin and C-TnT.GATA-4 and α-MHC expression were detected by RT-PCR.Results The cultured cells grew stablely and proliferated faster,mainly appeared to be fusiform and fibroblastic morphology.After induced by OT for one week,most cells became spindle-like and pillar-form with oval nuclei in the center of cells and similar to cardiomyocytes.The direction of the cell arraying was identical gradually.After four weeks,a majority of cells enlarged,lengthened,showed stick-like shape and paralleled closely.MSCs induced by OT could be identified by the positive staining for desmin,α-sarcomeric actin and C-TnT.Laser scanning confocal microscopy indicated that desmin（α-sarcomeric actin） appeared in red,cTnT was green.When they wereboth been showed,it changed to yellow.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.Conclusions MSCs can differentiate into cardiomyocytes in vitro after being induced by OT.