新生大鼠骨骼肌卫星细胞的原代培养及标记方法

本文用等密度沉降离心法分离出新生大鼠骨骼肌卫星细胞，观察其生长规律，检测分裂指数及生长曲线。用胶体金、DAPI及Brdu标记液分别标记细胞8、24、48h，测标记后细胞的生长曲线及标记率。结果表明分离所得肌卫星细胞纯度95％以上，梭形，培养第5天细胞排列出现方向性，并开始融合成肌管，第3—5天增殖旺盛，5代以内生长较快。三种标记方法对肌卫星细胞有不同程度毒性。胶体金标记法毒性最小，标记率100％；DAPI标记细胞率为100％；Brdu毒性最大且标记率仅为10％。本实验方法简单、快速，所得肌卫星细胞纯度高，性能好。胶体金标记法适合于电镜研究，DAPI标记法适合于光镜研究，Brdu标记法较差。

Primary culture and labeling method of skeletal satellite cells from neonatal rat

Density gradient centrifugation was used to isolate and purify skeletal satellite cell (SC). Satellite cells were cultured in M199 and growing state was recorded. Index of culture cell division and growth curve were detected. Cultured SCs were labeled by colloidal gold, DAPI and Brdu for 8, 24, 48 hours respectively. The labeling rate and growth curve after labeling were observed. SC purification was more than 95%. Shape of SC appears to be shuttle-like. At the 5th day, SCs arranged with a direction and begin to fuse myotubes. The cells proliferated very fast during day 3 to day 5 and grew very well for 5 passages. The different toxicity to SC was seen in three labeling methods. Among them, colloidal gold is the least toxic and its labeling rate was l00%. DAPI labeling rate was l00% too but had a little greater toxicity than colloidal gold method. Brdu method had the most serious toxicity and its labeling rate was on1y 10%. The method of isolating and culturing SC was simple and fast with nice purity. Labeling rate was high and toxicity was low for colloidal gold labeling method. Labeling rate was high and toxicity was middle for DAPI labeling method. The Brdu labeling method showed a high toxicity.