牦牛IGF2介导PI3K/AKT通路促进成纤维细胞增殖的机制研究

The insulin-like growth factor 2 (IGF2) as a candidate gene of yak molecular breeding, can regulating the body's growth, development and disease occurrence, and PI3K-ATK have played a vital function role in this process. Previously study showed that overexpression of yak IGF2 can promote fibroblast proliferation, but its mechanism is unclear. This project intends to construct the gene knocks out and overexpression vectors of yak IGF2, and transfection these two vectors into yak fibroblast for obtaining the knock out and overexpression of cell lines. The variation of key genes and proteins in IGFs and PI3K/AKT pathway was detected by confocal laser scanning microscope, flow cytometry and western blot. Transgenic mice model was constructed through nuclear transfer for verification the variation of the key genes and proteins in PI3K/AKT pathway. And which will demonstrate the mechanism of IGF2 mediated PI3K/AKT pathway to promote fibroblasts proliferation. This research will provide the theory basis for yak growth development and molecular breeding.

胰岛素样生长因子2（IGF2）作为牦牛分子育种的候选基因，可调控牦牛生长、发育及疾病的发生，且PI3K/Akt通路在此过程中发挥重要的功能。项目组成员前期研究发现过表达牦牛IGF2能促进成纤维细胞增殖，但其机制尚不清楚。本项目拟构建牦牛IGF2基因CRISPR/Cas9敲除和过表达载体转染牦牛成纤维细胞系后，获得其敲除和过表达细胞株，通过聚光共聚焦、流式细胞和免疫印迹等分子生物学方法检测IGFs和PI3K/AKT通路中关键基因和蛋白的表达及对细胞增殖的影响，核移植构建IGF2转基因小鼠模型，验证PI3K/AKT通路关键分子的变化，深入揭示IGF2介导PI3K/AKT通路促进成纤维细胞增殖的机制，为牦牛的生长发育和分子育种提供理论依据。

胰岛素样生长因子2（IGF2）作为牦牛分子育种的候选基因，可调控牦牛生长、发育及疾病的发生，且PI3K/Akt通路在此过程中发挥重要的功能。团队前期研究发现过表达牦牛IGF2能促进成纤维细胞增殖，但其机制尚不清楚。项目分析了牦牛各个组织部位中IGF2基因和蛋白的定位与表达。从细胞水平探究了IGF2介导PI3K/AKT通路促进成纤维细胞增殖的机制和异同。对PI3K/AKT信号通路中关键功能基因PI3KCG等基因进行表达定位和验证；解析了IGF2基因在牦牛成纤维细胞增殖和生殖轴系中的作用和调控机制。项目结果确定了IGF2作为牦牛分子育种的候选基因，可调控牦牛生长、发育和生殖；细胞水平解析了IGF2基因通过PIK3CG负反馈调节牦牛成纤维细胞增殖的分子机制。从组织水平验证了牦牛组织中IGF2负调节PIK3CG下游分子机制；团队发现IGF2和AR在生殖轴系协同响应HPG轴对性腺的激素调节，并在牦牛生殖系统调控和睾丸组织中发挥主要功能；探究发现IGF2通过AR在牦牛生殖轴系的发挥调节作用。 上述结果深入挖掘了IGF2在牦牛生长、发育和生殖过程中的作用和机制，为牦牛的生长发育和分子育种研究提供理论依据。