摘要
目的 探讨 5 -氮杂胞苷 ( 5 -Aza)诱导间充质干细胞 (MSCs)分化为成肌细胞的相关机制。 方法 分离纯化 4～ 6周龄Balb C小鼠骨髓MSCs,以不同浓度 5 -Aza作用 ,用四唑盐(MTT)法测定细胞生长活力 ;逆转录 -聚合酶链反应 (RT -PCR)鉴定肌形成调节因子 5 (Myf5 )、成肌素 (myogenin)表达 ;免疫组织化学染色鉴定结蛋白 (desmin)、肌凝蛋白 (myosin)表达 ;观察细胞形态变化。 结果 1,3μmol L 5 -Aza对细胞增殖无明显影响 ,2 0 μmol L 5 -Aza对细胞有毒性作用 ,影响其增殖。 10 μmol L 5 -Aza诱导后 6h ,MSCs表达Myf5 ,9h达最高 ;5 μmol L5 -Aza诱导后9h ,MSCs表达Myf5 ,12h达高峰。 10 μmol L5 -Aza诱导后 2 4h ,MSCs表达myogenin ,48h达最高 ;5 μmol L5 -Aza诱导后 6d ,MSCs表达myogenin ,且为高峰。 10 μmol L5 -Aza诱导后 7d ,部分细胞表达desmin ;14d部分细胞表达myosin。 10 μmol L5 -Aza诱导后 9d ,部分细胞胞体明显增粗 ,14～ 16d后可见有肌管样细胞出现。 结论 5 -Aza诱导MSCs向成肌细胞定向分化 ,可能是因其产生的去甲基化作用使MSCs中处于转录失活阶段的肌分化调控基因Myf5等得以表达 ,从而调控MSCs向肌细胞系定向分化并逐步表达myogenin ,最后分化为肌管样细胞的过程。
Objective To investigate the related mechanisms of mesenchymal stem cells (MSCs) differentiating into myoblasts induced by 5-azacytidine (5-Aza). Methods Bone marrow-derived MSCs of 4-6 weeks old mouse were separated and purified, and then given 5-Aza with different concentrations. The growth ability of cells was assayed with methyl thiazolyl tetrazolium method. The expressions of myogenic regulator factor 5 (Myf5) and myogenin were evaluated with reverse transcription-polymerase chain reaction method, the antigen expressions of desmin and myosin with immunohistochemistry method and the morphological changes observed. Results The cell proliferation was not affected by 1 μmol/L or 3 μmol/L of 5-Aza, but 20 μmol/L of 5-Aza induced the toxic effect on proliferation of MSCs. MSCs began to express Myf5 at the 6th hour and reached the highest at the 9th hour after induction by 10 μmol/L of 5-Aza. MSCs began to express Myf5 at the 9th hour and reached the highest at the 12th hour after induction by 5 μmol/L of 5-Aza. MSCs began to express myogenin at the 24th hour and up to the highest at the 48th hour after induction by 10 μmol/L of 5-Aza. After induced by 5 μmol/L of 5-Aza, MSCs began to express myogenin up to peak at the 6th day; some of MSCs expressed desmin at the 7th day and myosin at the 14th day. Some cells were obviously enlarged at the 9th day after induction. Myotube-like cells were found at the 14-16th days. Conclusions The reason that 5-Aza can induce orientational differentiation of MSCs into myoblasts is probably the methylation effect of 5-Aza, by which 5-Aza can induc the expression of myogenic regulatory factor Myf5 in inactive transcription and ever since regulate the course that MSCs orientationally differentiate into myoblasts, gradually express myogenin and finally differentiate into myotube-like cells.
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2003年第4期207-211,共5页
Chinese Journal of Trauma
基金
国家重点基础研究发展规划基金资助项目(G19990 5 42 0 5 )
