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目的:探索骨形态发生蛋白2(BMP-2)、P53抑制剂(PFT-α)以及二者联合诱导对大鼠骨髓间充质干细胞(BMSCs)向心肌细胞分化的影响。方法:分离4周龄SD大鼠的骨髓间充质干细胞并进行体外培养,对第二代BMSCs定向诱导,依次为BMP-2组、PFT-α组、BMP-2+PFT-α组以及空白对照组,相差显微镜观察细胞的形态学变化。免疫细胞化学法检测诱导4周后心肌特异性标记物(α-actin、CTn T、desmin、P38-Mark)的表达,并进行统计学分析;透射电镜观察细胞的内部结构。结果:诱导4周后,诱导组大鼠BMSCs的细胞形态变长,呈杆状,紧密平行排列,方向一致;空白对照组大鼠BMSCs细胞呈星形或三角形。免疫细胞化学检测显示:各诱导组BMSCs均可见α-actin、C-Tn T、desmin、P38-Mark阳性的细胞,其中联合诱导组的阳性表达率高于BMP-2或PFT-α单独诱导组,诱导各组与对照组的以上各标记物的阳性率具有统计学差异(P﹤0.05)。透射电镜结果显示:诱导组大鼠BMSCs细胞呈分支杆状,核卵圆形,且位于细胞中央,细胞质中有肌丝平行排列,可见线粒体、大量粗面内质网、核糖体等。结论:BMP-2、PFT-α均可诱导BMSCs向心肌样细胞分化,且两者联合应用效果强于单一应用的效果。
Objective: To explore the effects of bone morphogenetic protein 2 (BMP-2), P53 inhibitor (PFT-α) and their combination on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes. Methods: Bone marrow mesenchymal stem cells from 4-week-old SD rats were isolated and cultured in vitro. The second generation of BMSCs were induced by BMP-2, PFT-α, BMP-2 + PFT-α and blank Control group, phase contrast microscope to observe the morphological changes of cells. The expression of cardiac-specific markers (α-actin, CTn T, desmin, P38-Mark) were detected by immunocytochemistry after 4 weeks of induction and analyzed statistically. The internal structure of the cells was observed by transmission electron microscopy. RESULTS: After induction for 4 weeks, the morphology of BMSCs in the induction group became longer and rod-like, arranged in close parallel and in the same direction. The BMSCs in the blank control group showed star or triangle shape. The results of immunocytochemistry showed that the positive expression rates of α-actin, C-Tn T, desmin and P38-Mark in BMSCs were all higher than that in BMP-2 or PFT-α alone group , There was a significant difference (P <0.05) in the positive rate of the above markers in each group and the control group. Transmission electron microscopy showed that the BMSCs in the induction group were branched and nuclear-oval, and located in the center of the cells. The myofilaments were arranged in parallel in the cytoplasm, showing mitochondria, a large number of rough endoplasmic reticulum and ribosomes. CONCLUSION: BMP-2 and PFT-α can induce BMSCs to differentiate into cardiomyocyte-like cells, and the combined effect of them is stronger than that of single application.