大鼠肛提肌卫星细胞的分离纯化及鉴定

Separation, purification and identification of satellite cells from rat levator ani muscle

摘要:

目的： 探讨大鼠肛提肌卫星细胞分离和纯化方法，观察肛提肌卫星细胞的体外增殖和成肌特性。方法： 采用改进的胰蛋白酶和Ⅰ型胶原酶来消化分离大鼠的肛提肌卫星细胞，结合差速贴壁法进行纯化，从而获得高纯度的大鼠肛提肌卫星细胞。运用显微镜观察细胞的形态和生长状况；CCK-8检测体外培养的肛提肌卫星细胞生长情况并绘制生长曲线；免疫荧光法对不同时期的卫星细胞进行鉴定。结果： 显微镜下观察发现，分离出的大鼠肛提肌卫星细胞生长较好。CCK-8检测结果显示，体外培养的前1、2 d细胞生长缓慢，3 d以后细胞呈倍数扩增，第10天细胞生长速度下降，部分细胞出现分化。免疫荧光和DAPI染色显示不同时期的肌卫星细胞，其特征性基因表达有差异。结论： 本方法成功从大鼠肛提肌组织中分离出了具有增殖、分化能力的肛提肌卫星细胞，为应用肛提肌卫星细胞探讨肌肉的损伤、修复机制奠定了前期基础。

Abstract:

Objective: To explore a method for separation and purification of satellite cells (SCs) from rats levator ani muscle (LAM), and to observe the proliferation and myogenic characteristics of SCs in vitro. Methods: The SCs from rat LAM were separated by enzyme digestion with collagenase type 1 and trypsin, and purified by differential wall adhesion method. From that, we obtained highly purified SCs from rat LAM. The morphology and growth of primary and passage cells were observed by inverted microscope. CCK-8 method was used to detect the growth of LAM SCs (cultured in vitro) and draw the growth curve. Immunofluorescence was used to identify SCs in different stages. Results: Microscopically, it was observed that SCs separated from rat LAM grew well. In CCK-8 test, the SCs grew slowly in the first to second days of in vitro culture, after 3 days, the SCs augmented multiply. On the 10th day, the cell growth rate decreased and some cells began to differentiate. Immunofluorescence and DAPI staining showed that the characteristic gene expression of protein was observed different in different stages of SCs. Conclusion: In this study, SCs were successfully separated from rat LAM tissue with good proliferation and differentiation ability in vitro, laying a preliminary foundation for exploring the mechanism of muscle injury and repair by LAM SCs.