骨骼肌卫星细胞作为肌肉的前体干细胞，其分化的分子机理研究一直是生命科学研究领域的基本问题。前期工作发现，葡萄糖调节蛋白94（Gloucose requlated protein 94，Grp94）和磷脂酰肌醇3激酶相互作用蛋白1（PI3K interacting protein 1，PIK3IP1）的表达变化能够影响PI3K/Akt/mTOR信号通路及牛骨骼肌卫星细胞的分化，初步结果表明Grp94与PIK3IP1相互结合。本项目拟采用CRISPR/dCas9技术调控Grp94和PIK3IP1的表达，以激活剂和抑制剂调控PI3K/Akt/mTOR信号通路的表达，证明Grp94通过PI3K/Akt/mTOR信号通路影响细胞分化。进一步采用免疫共沉淀、荧光能量共振转移和蛋白质点突变技术阐明Grp94与PIK3IP1直接结合调节PI3K/Akt/mTOR信号通路及牛骨骼肌卫星细胞分化的分子机理。

Skeletal muscle satellite cell is a kind of muscle precursor stem cells. The molecular mechanism through which skeletal satellite muscle cell differentiates has been an important and basic question in the research field of life science. Our previous study revealed that, expression of Grp94 (Gloucose requlated protein 94) and PIK3IP1 (PI3K interacting protein 1) were associated with PI3K/Akt/mTOR signal pathway and bovine skeletal satellite muscle cell differentiation, furthermore Grp94 could interact with PIK3IP1. Thus, in this study, CRISPR/dCas9 will be used to regulate the expression of Grp94 and PIK3IP1, activator and inhibitor are used for regulating PI3K/Akt/mTOR signal pathway to prove Grp94 affect bovine skeletal satellite muscle cell differentiation through PI3K/Akt/mTOR signal pathway. Moreover, co-immunoprecipitation (Co-IP), fluorescence resonance energy transfer (FRET) and protein point mutation techniques will also be used to illustrate whether Grp94 regulates PI3K/Akt/mTOR signal pathway and bovine skeletal satellite muscle cell differentiation directly through PIK3IP1. Our findings would be helpful to reveal the molecular mechanism of bovine skeletal satellite muscle cell differentiation.