论文部分内容阅读
本研究的主要目的在于探明PI3K/Akt通路在肌细胞生脂转分化中的调控作用.试验培养并诱导C2C12肌细胞生脂转分化,同时使用抑制剂Wortmannin处理细胞抑制PI3K的激活,或者使用特异性siR NA转染沉默细胞内源PI3K基因的表达,观察其对肌细胞生脂转分化的影响.结果表明,随着C2C12细胞的生脂转分化,PI3K蛋白(P55亚基和P85亚基)和其下游效应分子Akt的磷酸化水平,在转分化前期提高而在转分化后期明显降低.使用Wortmannin处理细胞能够有效抑制PI3K/Akt激活,这导致C2C12细胞的生脂转分化明显受到抑制,细胞内脂肪生成量显著降低,生脂基因PPARγ、C/EBPα、FABP4和FATP1的表达水平均显著下调.使用特异性siR NA转染细胞显著下调PI3K基因表达水平和蛋白质含量,同样明显抑制了C2C12细胞的生脂转分化.此外,在转分化过程中抑制PI3K/Akt的活性和表达还激活了Caspase-3并导致细胞凋亡.综合上述结果可以确认PI3K/Akt的正常表达和激活是肌细胞生脂转分化必不可少的.
The main purpose of this study is to investigate the regulation of PI3K / Akt pathway in myogenic transdifferentiation of myocytes.Methods Cultured and induced adipogenic transdifferentiation of C2C12 myocytes, while the inhibitor of Wortmannin treatment inhibited PI3K activation, or use Specific siR NA transfection was used to detect the expression of endogenous PI3K gene and to observe its effect on adipogenic differentiation of myocytes.Results showed that PI3K protein (P55 subunit and P85 subunit ) And its downstream effector Akt phosphorylation increased in the early stage of transdifferentiation and decreased significantly in the late stage of transdifferentiation.Wortmannin treatment of cells could effectively inhibit the activation of PI3K / Akt, which led to the inhibition of adipogenic differentiation of C2C12 cells, The intracellular fat production was significantly decreased, and the expression levels of adipogenic genes PPARγ, C / EBPα, FABP4 and FATP1 were significantly down-regulated.Using specific siRNA transfected cells significantly downregulated the expression of PI3K gene and protein content, also significantly inhibited the expression of C2C12 In addition, inhibition of PI3K / Akt activity and expression during transdifferentiation also activates Caspase-3 and leads to apoptosis. Confirmed PI3K / Akt activation and expression of normal muscle cells are essential adipogenic transdifferentiation.