大鼠骨髓基质干细胞体外培养方法的实验研究

Method of mesenchymal stem cells culture in vitro

目的研究培养骨髓基质干细胞(MSCs)的简易方法和其在体外经5-氮胞苷诱导为心肌细胞,为心肌梗死后心力衰竭的干细胞移植治疗提供理想的种子细胞。方法采用传统的密度梯度离心分离加贴壁法和略加改进的Wakitani方法。具体操作是:取SD大鼠分离出股骨和胫骨,用10%DMEM培养基冲出骨髓液,以2×105/ml密度接种于培养瓶中,培养出的骨髓基质干细胞连续传3代,使细胞纯化后,用5-氮胞苷诱导,观察细胞形态改变。结果大鼠骨髓液接种于培养瓶中后,第2天大部分细胞已经贴壁,贴壁生长的细胞以分散、克隆集落的方式增殖,细胞大多呈梭形,7～10d后克隆集落逐渐增多,细胞融合,传代培养后细胞呈分布均匀的纺锤形细胞生长。MSCs经10!mol/L5-氮胞苷诱导3周后,发现细胞形态较前更加细长,呈梭形,类似心肌细胞样形态。结论应用略加改进的Wakitani方法,能较好地进行骨髓基质干细胞的培养和扩增,且操作简便;在体外培养的骨髓基质干细胞经诱导可转化为心肌细胞,有望成为心肌梗死后心力衰竭干细胞移植治疗的细胞材料。

Objective To evaluate the simple method for rat bone marrow mesenchymal stem cells （MSCs） culture and proliferation and generate into cardiomyocytes, providing potential therapeutic application for heart failure after myocardial infarction. Methods MSCs was isolated from adult rats using density gradient centrifugation and anchoring culure routinely. The other methods was operated as following： isolated thighbone and shankbone from a SD rat. The bone marrow cells were inoculated to tank farming in 2×10^5/ml density. Immortalized cells were obtained by frequent subculture for 3 era. After being co-cultured with 5-azacytidine, observe the morphologic change. Results The MSCs attached and grew as fibroblastic cells at 24 hours after initial planting. The hematopoietic stem cells that did not attach to the dish were washed from the culture with each medium changes. MSCs grew rapidly, developed into visible symmetric colonies at about 3 days and reached confluence at 7 to 10 days. While the cells were permitted to proliferate to confluence, MSCs were spindle-shaped morphology,small and arranged like pectinate.The MSCs did not differentiate spontaneously during culture expansion and maintained a normal MSCs biological features. After being co-culture with 5-azacytidine, some of MSCs became spindle-like cells,like cardiomyocytes- shaped. Conclusion MSCs can be cultured by the simplified Wakitani method. Cardiomyocytes can be generated from MSCs.It suggests that MSCs might be a new cell sources for stem cells transplantation in heart failure therapy.