大鼠骨骼肌细胞的原代培养及鉴定

探讨体外条件下骨骼肌卫星细胞的原代培养、纯化及鉴定的方法。取新生3～5 d的SD大鼠处死清洗后,采用Ⅰ型胶原酶、胰蛋白酶联合酶消化30 min,细胞悬液过滤离心后,用含有10%胎牛血清的培养液悬浮细胞,1 h后重新接种,重新接种2次,最后接种于L-多聚赖氨酸铺底的培养瓶中;采用骨骼肌肌动蛋白(α-sarcometric actin)免疫荧光方法鉴定骨骼肌细胞。肌卫星细胞培养5～6 h后开始贴壁,48 h完全贴壁,之后细胞进一步增多并相互融合,逐渐按一定方向呈有序排列。当细胞增殖至70%密度时,有融合趋势,加入分化培养基培养48 h后可形成多核的肌管,行免疫荧光染色,90%以上的细胞α-sarcometric actin染色阳性,证明培养的为骨骼肌细胞。

Primary Culture and Identification of Rat Skeletal Muscle Cells

This paper explored a reliable method for primary culture,purification and identification of satellite cells of rat skeletal muscles in vitro.New-born rats,3～5 days old,were obtained,the tissue pieces were digested in a mixture of type I collagenase and trypsin and then filtered through 100,200 and 400 mesh screens in order;the suspended cells were centrifuged,resuspended in a culture medium containing 10% fetal bovine serum(FBS) and inoculated into cell culture bottle and incubated;then the cell suspension in the culture bottle was transfered two times to new culture bottles,each time after one-hour interval;finally the cells were inoculated into culture bottle coated with L-polylysine.Skeletal muscle cells were identified by immunofluorescence method using α-sarcometric actin specific monoclonal antibody.The primary muscle satellite cells started adhering in 5～6 hours,completed adhering the wall within 48 hours.The cells further increased in number and fused mutually,and gradually assumed an ordered arrangement according to certain directions.The cells had the tendency to fuse after reaching 70% confluence.After 48 hours of addition of the differentiation culture medium,most of the cells formed multinucleated myotubes.Immunofluorescent staining shows more than 90% of the cells are positively stained for α-sarcometric actin,which proves that the obtained cells are skeletal muscle cells.