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目的研究骨髓间充质干细胞(MSCs)移植入放疗的mdx鼠后dystrophin/utrophin的表达变化。方法将BrdU标记的大鼠P5代MSCs移植入放疗处理的mdx鼠,分别于移植后4、8、12和16周断头处死取其腓肠肌,冰冻切片应用免疫荧光、RT-PCR及Western blotting检测BrdU/dystrophin及utrophin的表达变化。另分别取5只正常C57BL/6鼠和mdx鼠作阳性和阴性对照。结果经γ射线放疗预处理的mdx鼠,在尾静脉移植MSCs后4周时肌纤维抗肌萎缩蛋白的表达少于1%,随移植时间延长增加,16周时为15%,dystrophin/BrdU激光共聚焦免疫荧光显示肌细胞核中移现象较同龄mdx鼠对照组减少,边居增多。RT-PCR及Western blotting检测到dystrophin表达也随移植时间延长增多。移植后mdx鼠肌细胞膜utrophin表达较对照组mdx鼠减少,RT-PCR结果显示mRNA在移植前后没有明显改变。结论MSCs移植后mdx鼠的肌细胞膜有dystrophin表达,激光共聚焦技术表明dystrophin阳性肌纤维部分来自于移植的MSCs。utrophin在mdx鼠肌膜上表达上调,MSCs移植后dystrophin表达对utrophin有一定的抑制作用,两者存在互补关系。
Objective To investigate the expression of dystrophin / utrophin after bone marrow mesenchymal stem cells (MSCs) transplantation into mdx mice after radiotherapy. Methods BrdU-labeled rat P5 MSCs were transplanted into mdx mice treated with radiotherapy. The gastrocnemius muscles were harvested at 4, 8, 12 and 16 weeks after transplantation, respectively. Immunofluorescence, RT-PCR and Western blotting were used to detect frozen-thawed sections. BrdU / dystrophin and utrophin expression changes. Another five normal C57BL / 6 mice and mdx mice were taken as positive and negative controls. Results The mdx mice pretreated with γ-ray radiotherapy had less than 1% myofibrillar dystrophin expression at 4 weeks after transplantation of MSCs in the caudal vein, increased with the transplantation time and 15% at 16 weeks. The dystrophin / BrdU laser Focused immunofluorescence showed that the myocyte nuclear shift in the control group decreased compared with the same age mdx mice, increased living. Detection of dystrophin expression by RT-PCR and Western blotting also increased with transplantation time. After transplantation, the expression of utrophin in mdx muscle cells decreased compared with mdx mice in control group, and RT-PCR results showed that there was no significant change in mRNA before and after transplantation. Conclusion MSCs transplantation dystrophin expression in muscle cell membrane, laser confocal laser scanning microscopy shows dystrophin-positive muscle fibers derived from the transplanted MSCs. utrophin is up-regulated in the myofibroblasts of mdx mice, and the expression of dystrophin after MSCs transplantation has a certain inhibitory effect on utrophin, and there is a complementary relationship between them.