摘要目的 探讨通过条件培养液(CM)和高浓度血清即20%的胎牛血清(FBS)联合作用下,myocardin在骨髓来源的间充质干细胞向平滑肌细胞(SMC)定向分化过程中的表达特点.方法 采用伞骨髓贴壁法分离骨髓问充质干细胞,CM联合20%FBS诱导骨髓间充质干细胞,同时设立持续10%FBS、单20%FBS及单CM诱导下的骨髓间充质十细胞对照组和SMC阳性对照组,分别于诱导前及诱导3、7、10和14 d时观察细胞形态的变化,并在相应的时间点用免疫荧光法、逆转录聚合酶链反应法、Western blot半定量分析法榆测myocardin以及SMC表面各种标志基因的表达变化,用透射电镜检测诱导后细胞内肌丝存在以此来证实诱导分化成功.结果 光镜观察显示诱导前至诱导3、7、10和14 d时,骨髓问允质十细胞南纺锤形、多角形、扁平形和小圆形等多种形态逐渐转变为单一的长梭形,在诱导21 d后显示出明显的峰-谷特征.免疫荧光结果显示表达myocardin的阳性细胞数量逐渐增多,表达部位由胞质逐渐移向胞核,而表达平滑肌标志基因Ot一平滑肌肌动蛋白(Ot.SMA)、平滑肌肌动蛋白重链(SM-MHC)的细胞也随之增加,同时联合诱导后3、7、lO和14 d表达myocardin和平滑肌标志基因(α-SMA或SM-MHC)的双阳性细胞数最逐渐增加.RT-PCR结果显示myocardin的mRNA表达逐渐上调,在诱导7 d达到高峰后表达基本稳定(P<0.05);平滑肌标志基因α-SMA及SM22α的表达随着myocardin表达上调而增加(r=O.81,P<0.05),诱导10 d后表达基本稳定.Western blot结果显示myocardin蛋白和平滑肌标志基因α-SMA蛋白表达量在诱导后明显增加(r=0.84,P<0.05).透射电镜结果显示诱导21 d后的分化细胞中有肌丝的存在.在不同时间点各组之间的差异有统计学意义(P<0.05).结论 CM联合高浓度的血清可有效诱导骨髓间充质干细胞向SMC分化,myocardin在此分化过程中起了重要作用.骨髓来源的间充质干细胞可以在诱导下定向分化成为SMC.

abstractsObjective To investigate the expression profiles of myoeardin gene during the differentiation of bone marrow-derived mesenchymal stem cell to smooth muscle cells in the conditional medium combiued with a high concentration of fetal bovine serum (FBS). Methods Marrow-derived mesenchymal stem cells were isolated and purified from mouse femoral bone and shinbones using differential adherent methods. Cells at the third passage were induced by 20% FBS in conditioned medium, conditioned medium alone, 20% FBS or 10% FBS alone respectively. Mouse aortic smooth muscle cells were cultured as the positive control. Levels of mRNA and protein expression of myoeardin and several smooth muscle cells marker genes were determined by immumofluorescenee, RT-PCR and Western blot before and 3, 7, 10, 14d after the induction. The presence of smooth muscle myofilaments was detected by using transmission electron microscope. Results Naive bone marrow-derivod mesenchymal stem ceils displayed multiple morphological forms including fusiform, polygon, oval, and micro-spherical, as compared to the single macro-spindle form after the induction. Typical appearance of peak valley was displayed on the 21st day after induction. At the same time, the expression of smooth muscle marker genes was reinforced along with an up-regulation of myocardin expression, lmmunofluoreecence study showed that the cells expressing myocardin and smooth muscle marker genes such as α-SMA and SM-MHC increased. Fluorescence domain of myocardin translocated from cytoplasm to nucleus and the amounts of double positive cells for myecardin with α-SMA or SM-MHC also increased. RT-PCR confirmed that the mRNA expression of myocardin increased gradually and remained stabilized after achieving its peak on the 7th day after induction. The expression of smooth muscle marker genes, α-SMA and SM22α, remained stable on the 10th day of induction. It was also confirmed by Western blot that the protein expression of both myocardin and α-SMA were markedly increased during the induction. Finally, transmission electron microscopy revealed the presence of mynfilament on the 21st day after induction. Conclusions Bone marrow-derived mesenchymal stem cells can be effectively induced into smooth muscle-like cells by conditioned medium combined with 20% FBS. Myocardin plays an important role in the differentiation process of bone marrow-derived mesenchymal stem cells to the peripheral smooth muscle cells.