经micro-dystrophin基因修饰后的自体骨髓间充质干细胞移植治疗mdx鼠的实验研究

目的探讨携带micro-dystrophin基因的自体骨髓间充质干细胞(mMSCs)在移植鼠体内分化为有功能肌细胞的可能性。方法采用逆转录病毒介导micro-dystrophin基因转染mdx小鼠MSCs(mMSCs),通过尾静脉注射移植治疗mdx鼠,在移植后不同时间点并检测血清激酶(CK)值,对腓肠肌进行HE染色、计数核中心移位纤维(CNF)比例,并用免疫荧光法,、逆转录-聚合酶链反应、Westernblot检测测micro-dystrophin的表达。结果移植后各时间点血CK值下降,腓肠肌病理改变较对照组有所改善,CNF比例下降,差异有统计学意义,部分肌细胞膜能表达micro-dystrophin蛋白,并随移植时间延长micro-dys-trophin阳性肌纤维比例增加,分别达到1%(8周时)、7%(12周时)和15%(16周时)。RT-PCR和Westernblot也发现,随着移植时间的延长,micro-dystrophin表达增加。结论自体mMSCs可携带外源性micro-dystrophin基因在受体鼠体内分化为micro-dystrophin阳性肌细胞。

The Study on the Treatment of Micro-dystrophin Gene Modified Autologous Bone Mesenchymal Stems Cells Transplantation on Mdx Mice

Objective To study the possibility of genetically modified autologous mesenchymal stem cells differentiated into functional muscle cells in transplanted mdx mice. Methods mMSCs were infected with retrovirus containing micro-dystrophin and transplanted into mdx mice through tail vein. After transplantation, at each time point, the level of blood cream kinase (CK) was detected. After HE staining, morphology of myocytes was observed and the rate of cent rally nucleatedfibers ( CNF ) was calculated. Expression of micro-dystrophin was detected using the method of immunofluorescence, RT-PCR and Western-blot. Results After transplantation, CK was deceased. The pathological change in muscles was improved and the proportion of CNF was decreased. Compared with non-transplanted mice, the CNF rate in the gastrocnemius of mice was reduced statistically. Overtime, the micro-dystrophin positive fibers increased 1% ( at 8 weeks), 7%(at 12 weeks) , 15% (at 16 weeks) in the gastrocnemius respectively. By RT-PCR and Westernblot, the micro-dystrophyin expression also increased as time going. Conclusion The genetically modified autologous MSCs by micro-dsytrophin gene could differentiate into micro-dystrophin positive cells in transplanted mdx mice.