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目的:观察骨髓间充质干细胞移植治疗迪谢内肌营养不良模型鼠后肌肉病理改变,及迪谢内肌营养不良致病基因dystrophin蛋白表达变化。方法:实验于2003-07/2004-07在华中科技大学同济医学院同济医院神经科实验室进行。取4～6周龄C57BL/6雄性小鼠的股骨和胫骨,采用密度梯度分离和贴壁培养相结合的方法分离培养骨髓间充质干细胞。①正常组:2周龄C57BL/6小鼠4只,不干预。②对照组:2周龄mdx鼠4只,左后腿腓肠肌注射50μL磷酸盐缓冲液。③移植组:2周龄mdx鼠4只,左后腿腓肠肌注射50μL骨髓间充质干细胞细胞悬液(约1×106)。16周后3组小鼠麻醉下取左腿腓肠肌中部肌肉,苏木精-伊红染色后观察核内移肌纤维占总肌纤维百分率,及dystrophin免疫组化染色计算阳性纤维与总肌纤维百分率。结果:12只小鼠均进入结果分析。①核内移肌纤维占总肌纤维百分率:正常组肌纤维少核内移(1.2%);对照组肌纤维大小不等,大量核内移(75%);移植组肌纤维核内移明显减少(23.3%)。②dystrophin染色阳性纤维与总肌纤维百分率:正常组为100%,移植组显著高于对照组(75%,23.3%)。结论:骨髓间充质干细胞移植治疗迪谢内肌营养不良模型小鼠mdx,可使其骨骼肌细胞膜骨架蛋白dystrophin恢复,肌肉病理改变好转,且疗效持续到治疗后4个月以上,提示骨髓间充质干细胞移植对mdx小鼠治疗有效。
OBJECTIVE: To observe the changes of muscular pathology and the expression of dystrophin protein in dystrophic muscular dystrophy (DM) after transplantation of bone marrow mesenchymal stem cells (BMSCs) in Dystrophin muscular dystrophy rats. Methods: The experiment was performed at the Neurology Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from July 2003 to July 2004. The femurs and tibias of C57BL / 6 male mice from 4 to 6 weeks old were collected and cultured. The bone marrow mesenchymal stem cells were isolated and cultured by density gradient and adherent culture. ① normal group: 2 weeks old C57BL / 6 mice 4, no intervention. ② Control group: 4 mdx mice of 2 weeks old, 50 μL of phosphate buffered saline of the left hind leg gastrocnemius. Transplantation group: Four mdx mice of 2 weeks old were injected with 50 μL bone marrow mesenchymal stem cell suspension (about 1 × 106) into gastrocnemius of the left hind leg. After 16 weeks, the muscles of the left leg gastrocnemius muscle were removed under anesthesia. The percentage of total muscle fibers in the nucleus pulposus was determined by hematoxylin-eosin staining, and the percentage of positive fibers and total muscle fibers by dystrophin immunohistochemical staining. Results: All 12 mice entered the result analysis. ① The percentage of intramuscular myofibers in the nucleus was less than that in the normal group (1.2%). The size of the muscle fiber in the control group was large (75%), and the number of intramuscular nuclei was significantly decreased in the transplanted group (23.3%) . ② The percentage of positive staining fibers and total muscle fibers of dystrophin staining in normal group was 100%, which was significantly higher in transplantation group than in control group (75%, 23.3%). CONCLUSION: The transplantation of bone marrow-derived mesenchymal stem cells in mice with dystrophic muscular dystrophy model can recover the dystrophin of skeletal muscle cells and improve the pathological changes of muscle, and the curative effect continues for more than 4 months after treatment, suggesting that bone marrow Mesenchymal stem cell transplantation is effective in the treatment of mdx mice.