骨髓干细胞移植后mdx鼠腓肠肌病理及Dystrophin的表达变化

Change of Morphology and Dystrophin Expression in mdx mice after Bone Marrow Stem Cells Transplantation

【目的】研究骨髓干细胞移植后mdx鼠腓肠肌组织病理变化及dystrophin的动态表达变化。【方法】7～9周mdx鼠20只平均分为4组,放疗后移植1.2×107个/只同种异基因全骨髓干细胞,于移植后4周、8周、12周及16周应用HE染色观察细胞形态及核中心移位(CNF),免疫组化及Westernblot方法检测dystrophin表达变化,C57鼠和未治疗mdx鼠各5只作阳性和阴性对照。【结果】C57鼠腓肠肌横切面可见肌细胞大小形态基本一致,无核中移现象。各细胞移植治疗组和对照组mdx鼠均有大量的炎细胞浸润,核中心移位明显。未治疗mdx鼠CNF最高,可达70%左右,移植后4周、12周和16周,CNF比例分别为55%、50%和44%。免疫荧光结果C57鼠肌膜呈完整的网状绿色荧光,mdx鼠肌膜基本未见绿色荧光;移植后4周肌膜dystrophin阳性纤维数大约占细胞总数的1%,随时间延长表达渐渐增多,8周、12周和16周时阳性细胞数分别占细胞总数的5%、10%和15%。Westernblot结果mdx鼠无dystrophin的表达,野生型C57鼠表达量最多,移植后4周mdx鼠仅见微弱表达,随时间延长表达量渐增,移植后16周表达量较移植8周明显增加。【结论】骨髓干细胞移植后mdx鼠腓肠肌CNF随移植时间延长逐渐减少,dystrophin的表达随时间延长增多,提示骨髓干细胞移植后长久持续参与受损骨骼肌的修复与再生。

[Objective] To investigate the change of morphology and dystrophin expression in mdx mice after the bone marrow stem cells transplantation. [Methods ] Twenty mdx mice （7-9 weeks old） were divided into 4 groups and bone marrow stem cells of C57 mice （6 to 8 weeks age） were injected intravenously into the mdx mice （7 to 9 weeks age）,which were preconditioned with 7 Gy γ-ray. Morphology and centrally nucleated fibers were observed by HE stain, the rate of CNF was calculated. Dystrophin expression was detected in Gastrocnemius muscle using immunofluorescence and Western blot at 4 weeks, 8 weeks, 12 weeks and 16 weeks after transplantation, respectively. 5 C57 mice and mdx mice acted as positive and negative controls indivially. [Results] The myocytes of normal C57 mice is polygon and uniform in size. Nuclei are localized borderline of myocytes. No inflammation was found in intracellular substance. The myocytes of treated and untreated mdx mice were round in shape and various in size. The obvious abnormality was nucleus centralized. The rate of CNF in mdx mice was highest, up to 70%. The rate of CNF became lower than that of untreated mdx mice after 12 weeks and 16 weeks transplantation, decreased to 50% and 44%. The skeletal muscular membrane of C57 mice was stained strongly green fluorescein. There was no green fluorescein in mdx mice. At four weeks after MSCs transplantation, less than 1% of the myofibres expressed dystrophin.At 16 weeks after transplantation,as many as 16% of the muscle fibres within an individual mouse expressed dystrophin. No dystrophin was detected in mdx mice by Western blot. At 4 weeks after transplantation, the dystrophin expression in mdx mice were detected very low, but it expressed increasingly over time, at 16 weeks expressed much more than those of 8 weeks . [Conclusion] The allogenic bone marrow stem cells which were injected into mdx mice have a trend to reach the injured muscle tissues and differentiate to fibers which can express dystrophin and the expression increased over time. It indicated that the bone marrow stem cells can participate in the repair and regeneration of the injured tissues permanently and constantly.