生长激素基因的克隆和表达及其对贵州地方黄牛骨骼肌细胞增殖的影响

Cloning and expression of the growth hormone gene and its effect on the proliferation of skeletal muscle cells of Guizhou cattle

摘要:

本研究旨在探究生长激素 (Growth hormone，GH) 对贵州地方黄牛骨骼肌细胞增殖的表达调控，探明超表达GH基因对骨骼肌细胞增殖的影响。首先利用反转录PCR扩增黄牛GH基因的蛋白质编码区 (Coding sequence，CDS)，将其克隆至pUCM-T载体，并连接转化构建超表达载体pEGFP-N3-GH。同时使用实时荧光定量PCR检测GH基因在贵州地方黄牛骨骼肌相关组织 (腰大肌与背最长肌) 中的表达情况，然后培养牛原代骨骼肌细胞并进行鉴定，并将GH基因超表达载体导入细胞以研究GH基因对牛骨骼肌细胞增殖以及骨骼肌生长发育相关因子胰岛素样生长因子-1 (Insulin like growth factor 1，IGF-1) 与胰岛素样生长因子-2 (Insulin like growth factor 2，IGF-2)基因表达的影响。实时荧光定量PCR结果显示，GH基因在贵州地方黄牛腰大肌中的表达量均高于背最长肌，其中在关岭牛和威宁牛腰大肌中的表达量显著高于背最长肌 (P<0.05)。细胞转染及增殖结果表明，相比于pEGFP-N3，pEGFP-N3-GH能极显著提高GH与IGF-1、IGF-2基因在骨骼肌细胞中的表达量，且在被检测的4个时期(6 h、12 h、24 h、48 h)，超表达GH基因组也能够极显著地提高骨骼肌细胞的增殖速率 (P＜0.01)。结果提示，GH基因可促进贵州地方黄牛骨骼肌细胞的增殖，对其具有正向的调控作用，这为进一步探究GH基因对贵州地方黄牛生长发育的影响机制奠定基础。

Abstract:

The aim of this study was to investigate the expression of growth hormone (GH) gene on skeletal muscle cell proliferation of Guizhou cattle. The coding sequence of cattle GH gene was amplified by reverse transcription PCR, cloned into the pUCM-T vector and then used to construct the GH gene overexpression vector pEGFP-N3-GH. The expression of the GH gene in skeletal muscle-related tissues (psoas major and longissimus dorsi) of Guizhou cattle was determined by real-time fluorescent quantitative PCR (RT-qPCR). This was followed by culturing and identification of the bovine primary skeletal muscle cells. Subsequently, we introduced the GH gene overexpression vector into the cells to investigate its effect on the proliferation of bovine skeletal muscle cells and the expression of insulin like growth factor 1 and 2 genes related to skeletal muscle growth and development. RT-qPCR results showed that the expression level of GH gene was higher in the psoas major than in the longissimus dorsi of Guizhou cattle, and the expression level in the psoas major of Guanling cattle and Weining cattle was significantly higher than in the longissimus dorsi (P<0.05). The transfection and proliferation results showed that pEGFP-N3-GH significantly increased the expression of GH, IGF-1, and IGF-2 genes in skeletal muscle cells compared to pEGFP-N3 (P<0.05), and that overexpression of the GH gene also significantly increased the proliferation rate of skeletal muscle cells at the four periods examined (P<0.01). Our results suggest that GH gene can promote the proliferation of skeletal muscle cells of Guizhou cattle and exerts a positive regulatory effect. This lays the foundation for further exploring the mechanism by which the GH gene affects the growth and development of Guizhou cattle.