改良法体外培养大鼠成肌细胞的实验研究

目的:探索一种新型体外分离培养新生大鼠骨骼肌成肌细胞的途径.方法:取新生Wistar大鼠的四肢骨骼肌适量,采用混合酶一步消化分离获取成肌细胞,将差速贴壁法和胰酶消化法相结合进行纯化,观察分离纯化成肌细胞的形态学特点、生长状况,绘制细胞生长曲线,利用扫描电镜观察成肌细胞表面结构并进行结蛋白免疫细胞化学特异性鉴定.结果:采用改良方法分离成肌细胞产量高、耗时短;在培养过程中双重纯化获取的细胞生长状态良好,接种后3～5 d增殖达高峰;在扫描电镜下可观察到成肌细胞膜表面有微绒毛突起;体外培养的成肌细胞中94%的细胞胞质呈结蛋白抗体染色强阳性.结论:改良方法分离纯化大鼠骨骼肌成肌细胞效果理想,是成肌细胞体外培养的有效途径之一.

Experimental study of rat myoblast cultured with an in vitro modified method

Objective:To explore a novel method for isolating and culturing neonatal rat myoblasts in vitro.Methods: Skeletal muscle from limbs of neonatal Wistar rats was obtained,from which neonatal rat myoblasts were isolated and harvested by direct mixed enzyme digestion.Then the cells were further purified by combination of differential velocity adherent and trypsinization methods.The morphologic property and growth state of the myoblasts were observed,the myoblasts growth curves were drawn,the superficial structure of myoblasts was observed by SEM and the specificity of cultured myoblasts was identified by anti-desmin immunocytochemistry.Results: Compared with the traditional method,the novel method produced more myoblasts while took less time.The cells harvested with double purification method proliferated well and reached the proliferative peak within 3-5 days after inoculation.Some microvillous prominences on cell membrane surface of myoblasts were found under SEM.Immunocytochemical examination showed that the cytoplasm of 94% of the cultured myoblasts was strongly positive by anti-desmin staining.Conclusion:The modified method for isolating and purifying myoblasts can give ideal result,and is an effective way to culture myoblasts in vitro.