山羊胎儿肌肉干细胞的分离培养与成肌诱导分化

Isolation, Culture and Myogenic Differentiation of Muscle Stem Cells in Goat Fetal

【目的】通过体外建立安淮山羊胎儿肌肉干细胞分离培养及成肌诱导分化的方法,为进一步研究调控山羊肌肉干细胞增殖与分化的分子机制提供实验材料。【方法】本试验选取山羊胎儿背最长肌肌肉组织,用眼科剪剪成肉糜后,用0.1%的Ⅰ型胶原酶消化40 min,然后利用0.25%的胰酶消化15 min。分离得到的细胞用生长培养基(20%FBS+80%DMEM/F12+青链霉素)培养于37℃、5%CO2培养箱内。培养2h后采用差速贴壁技术对细胞进行纯化,又2h后,重复纯化一次。待细胞生长至70%左右密度时可进行传代培养。每次传代培养均采用差速贴壁30min的方法进一步纯化肌肉干细胞,共纯化至第6代。利用免疫荧光技术检测第6代细胞中肌肉干细胞标记基因Pax7、Myo D1的蛋白表达情况,从而对分离得到的细胞进行鉴定。当肌肉干细胞生长至70%左右密度时,将生长培养基更换为分化培养基(2%FBS+98%DMEM/F12+青链霉素),诱导细胞向成肌方向分化并观察细胞的形态学变化。细胞诱导分化1d后,采用免疫荧光技术检测肌肉干细胞的分化标记基因Myog的蛋白表达情况。另外,分别提取诱导0、1、3、5、7d后的细胞的总RNA,通过反转录试剂盒反转成c DNA后,利用q PCR测定Myo D1和Myog的相对表达量。【结果】分离得到的细胞呈贴壁生长,其形态趋于稳定后主要呈长梭形或纺锤形。免疫荧光技术检测的第6代细胞中Pax7和Myo D1蛋白均为阳性表达。采用分化培养基诱导细胞分化后,在显微镜下可观察到随着诱导天数的增加,细胞开始分化、相互融合成肌管且具有一定的方向性。免疫荧光检测结果表明Myog蛋白呈明显的阳性表达。另外,q PCR结果显示,标志基因Myo D1和Myog均有表达,且Myo D1的相对表达量在分化的第1天相比于0天显著升高并维持到第3天,第5、7天开始显著下降但仍显著高于增殖期。Myog在分化不同天数的细胞中的相对表达量具有类似的趋向。【结论】分离得到了纯度较高的安淮山羊胎儿肌肉干细胞,且诱导后展现出较好的成肌潜力。研究结果可为进一步开展肌肉干细胞成肌分化的分子机制研究提供材料来源。

【Objective】 To establish the method for isolation, culture, identification and myogenic differentiation of fetalmuscle stem cells from Anhuai goat in vitro, and to provide experimental materials for further research on the molecular mechanism of goat muscle stem cell proliferation and differentiation. 【Method】 In this study, the goat fetal longissimus muscle tissue was selected and cut into meat emulsion with ophthalmology, digested with 0.1% type I collagenase for 40 min and then digested with 0.25% trypsin for 15 min. The isolated cells were cultured in growth medium（20% FBS ＋ 80% DMEM/F12 ＋ Penicillin） in a 37℃, 5% CO2 incubator. After culturing for 2 h, the cells were purified by differential adherent technique. After 2 h, the cells were purified again. The cells were subcultured when they reached 70% density. 30 min adherent method was used for further purification with each subculture of muscle stem cells until the sixth passages. Muscle stem cell marker genes Pax7 and Myo D1 were detected with the purified cells for identification. When muscle stem cells grew to a density of about 70%, the growth medium was displaced with differentiation medium（2% FBS ＋ 98% DMEM/F12 ＋ Penicillin）for myoblasts induction and the morphology of the cells was monitored. One day after the induction of the cells, the marker protein of muscle stem cells Myog was detected. In addition, total RNAs of cells induced at 0, 1, 3, 5, and 7 days were separately extracted and their relative expression amounts of Myo D1 and Myog genes were measured by q PCR. 【Result】 Isolated cells showed adherent growth, and their morphology tended to be long spindle after stabilization. Pax7 and Myo D1 expression were detected in the 6 th passage cells by immunofluorescence. After induction by differentiated medium, the cells started to differentiate and fuse with each other into myotubes with a certain directionality as the induction prolonged. Myog protein was detected by immunofluorescence assay. Differentiation marker genes Myo D1 and Myog were detected by q PCR. Expression of Myo D1 could be detected in the first day of induction and maintained until the 3 rd day, and its level began to decline from the 5 th day but still significantly higher than the proliferative phase. A similar dynamic was observed with the relative expression level of Myog in the differentiating cells.【Conclusion】In this experiment, the fetal muscle stem cells of Anhuai goat was obtained with high purity, which showed good myogenic potential after induction. The results provide material for further research on the mechanisms of myogenic differentiation of muscle stem cells.