胎鼠骨骼肌卫星细胞的原代培养、纯化与鉴定

Primary culture, purification and identification of skeletal muscle satellite cells from fetal rat

目的探讨胎鼠骨骼肌卫星细胞的原代培养、纯化与鉴定的方法，为胎儿和早产儿骨骼肌发育程序化的研究提供实验基础。方法Sprague—Dawley大鼠孕18d时剖宫取出胎鼠，手术显微镜下取胎鼠四肢骨骼肌肌肉，采用I型胶原酶和胰蛋白酶两步消化法分离胎鼠骨骼肌卫星细胞，差速贴壁法进行纯化。原代培养的生长培养基为（DMEM／F12＋20％FBS＋10％HS＋5ng／mlbFGF）。分化培养基为（DMEM／F12＋2％HS）。CCK-8法绘制胎鼠骨骼肌卫星细胞生长曲线图，细胞免疫组化染色鉴定纯化后细胞的肌源性标志蛋白Desmin，观察分化培养基条件下骨骼肌卫星细胞的成肌分化特性。结果胎鼠原代骨骼肌卫星细胞在培养的第3天进入对数生长期，5～6d达增殖平台期；纯化后的细胞90％以上表达结蛋白（Desmin）。在分化培养基的诱导下细胞之间相互融合形成具有自发节律性收缩特性的多核肌管。结论采用消化法和差速贴壁法分离纯化胎鼠骨骼肌卫星细胞，能够获得高活性和高纯度的具有成肌分化能力的骨骼肌卫星细胞。

Objective The aims of this study were to establish a reliable method for primary culture, purification and identification of skeletal muscle satellite cells from fetal rat in vitro and to provide experiment means for investigating fetal and preterm skeletal muscle developmental programming. Methods Fetal rats were obtained by caesarean section on day 18 of gestation. The limb skeletal muscles were isolated from fetal rats under the surgical microscope, disassociated with type I collagenase and trypsin by two-steps digestion method, and purified via differential adhesion. Growth medium contained 70% DMEM/F12ham, 20% FBS, 10% HS and 5ng/ml bFGF. Differentiation medium contained 98% DMEM! F12ham and 2% HS. CCK-8 assay was used to record fetal rat skeletal muscle satellite cells growth curves. The expression of desmin was identified by immunocytochemistry staining. The differentiation of satellite cells was observed by inverted microscopy. Results Fetal rat skeletal muscle satellite cells entered the logarithmic growth period after 3 d of culture in vitro, and the platform phase was on day 5 to day 6. The positive rate of desmin was beyond 90%. Cells could fuse with each other and form myotubes with rhythmic contraction phenomenon when cultured in the differentiation medium. Conclusion High purity and viability primary fetal rat skeletal muscle satellite cells with potential of myogenic differentiation could be isolated and purified by digestion and preplate technique.