FGF10基因调节山羊成肌细胞分化相关基因表达的研究

Study of FGF10 gene regulation on the expression of differentiation related genes in goat myoblasts

为了阐明FGF10基因对山羊成肌细胞分化的影响,试验以简州大耳山羊羔羊的成肌细胞为研究对象,体外培养并诱导分化为肌管,采用实时荧光定量PCR技术检测FGF10基因在诱导分化过程中的表达水平;同时采用腺病毒过表达和SiRNA干扰技术、形态学观察和实时荧光定量PCR技术检测FGF10基因功能获得和缺失对成肌细胞中肌管形成的影响,以及分化相关基因MyoD1、Myf5、Myf6、MyoG的表达情况。结果表明:成肌细胞在分化第2~4天时,出现大量肌管,诱导分化成功。FGF10基因随着成肌细胞分化时间的延长呈现先升高后下降的趋势,且在诱导分化第2天时极显著高于分化前(P<0.01)。过表达FGF10基因时成肌细胞中的肌管形成减少,极显著下调MyoD1(P<0.01)和显著下调Myf5(P<0.05)基因的相对表达水平;而干扰FGF10基因时成肌细胞中的肌管形成增加,极显著上调MyoG(P<0.01)和显著上调Myf5(P<0.05)基因的相对表达水平。说明FGF10基因可能是山羊成肌细胞分化的负调控因子。

To elucidate the effect of FGF10 gene on the differentiation of goat myoblasts, the myoblasts of Jianzhou big ear sheep were used as the research object. Myoblasts were cultured in vitro and induced to differentiate into myotubes. The expression level of FGF10 gene during the induced differentiation was detected by real-time quantitative PCR. Meanwhile, adenovirus overexpression and siRNA interference techniques, morphological observation and real-time quantitative PCR were used to detect the effects of FGF10 gene function acquisition and loss on myotube formation in myoblasts, as well as the expression of differentiation-related genes MyoD1, Myf5, Myf6 and MyoG. The results showed that a large number of myotubes appeared on the 2 nd, 3 rd and 4 th day of cell differentiation, and the induced differentiation was successful. FGF10 gene increased at first and then decreased with the continuation of differentiation time of goat myoblasts, and it was significantly higher during myogenic differentiation than it had been before differentiation on the 2 nd day(P<0.01). Overexpression of FGF10 decreased myotube formation and significantly down-regulated the mRNA expression levels of MyoD1(P<0.01) and Myf5 genes(P<0.05). Interference with FGF10 increased myotube formation and significantly up-regulated the relative expression levels of MyoG(P<0.01) and Myf5 genes(P<0.05). The results showed that FGF10 might be a negative regulator of goat myoblast differentiation.