TSP-1/CD36基因表达对绵羊卵泡发育的影响研究

Thrombospondin-1 (TSP-1) and its receptor CD36 are expressed in a variety of cells, including granular cells. These are involved in the inhibition of angiogenesis, lipid metabolism, immune response and other biological functions. Studies revealed that TSP-1 and CD36 knockout mice significantly improve the number of follicles. Exogenous hormone treatment in lambs that are one month of age leads to a ten-fold increase in the number of developed follicles compared to adult sheep. We compared hormone-induced development of the follicular granulosa cell transcriptome in lambs and adult sheep, and found that TSP-1 and CD36 expressions were significantly decreased in lambs. However, the changes were not significant in sheep. This project is intended as an in vitro culture of sheep granulosa cells, in which we analyse the effect of FSH on TSP and CD36 gene expression. The purpose is to either inhibit or activate the expression of TSP-1/CD36 in the cells, and to analyze the signaling pathways that affect the expression of downstream genes. We aim to ascertain the effect of TSP-1 expression on the number of developed follicles in sheep via in vivo injection of either activator or inhibitor. Through this project, we aim to clarify the molecular mechanism encoded by the TSP-1/CD36 gene in follicular development of sheep and its resulting effect on the number of follicles.

凝血酶敏感蛋白1(TSP-1)及其受体CD36在包括颗粒细胞的多种细胞中表达，参与抑制血管发生、脂代谢和免疫反应等生物学功能。有研究表明TSP-1和CD36基因敲除小鼠卵泡发育数显著提高。外源激素处理一月龄羔羊卵泡发育数可超出成年羊10倍左右。我们比对羔羊和成年羊激素诱导发育卵泡颗粒细胞转录组时发现：羔羊TSP-1/CD36的表达显著下降，而成年羊变化则不显著。我们推测羔羊颗粒细胞中通过TSP-1/CD36基因表达下调的信号通路而导致其卵泡超数发育。本项目拟通过体外培养绵羊颗粒细胞，分析FSH对TSP/CD36基因表达的影响；抑制或激活细胞内TSP-1/CD36的表达，分析所影响的其上下游基因表达的信号通路。通过体内注射激活剂或抑制剂，鉴定TSP-1/CD36的表达对绵羊卵泡发育数的影响。通过本项目研究，揭示TSP-1/CD36基因在绵羊卵泡发育过程中的分子调控机制及对卵泡发育数的影响。

我们以中国美利奴（新疆军垦型）成年母羊卵泡及卵巢颗粒细胞为试验材料，在GCs中探讨FSH影响TSP-1表达以及其调控的具体信号通路，分析TSP-1对卵泡激素合成、类固醇通路基因和GCs凋亡等生物学过程的分子调控机制；进一步通过体内注射TSP-1模拟肽分析TSP-1对卵泡发育和卵巢形态学变化的影响。结果表明:（1）PCR扩增从绵羊卵巢中获得了TSP-1和CD36基因的cDNA全长序列；TSP-1在肝脏、肺脏中高表达，CD36在心脏和卵巢中高表达。随着卵泡直径的增大，TSP-1表达量逐渐降低，CD36 mRNA在中等大小卵泡中高表达；TSP-1和CD36在GCs中表达量均显著高于卵母细胞和卵丘细胞。（2）成功构建了重组表达质粒 pET19b-TSP-1/CD36和获得了TSP-1/CD36抗体；在初级卵泡、次级卵泡和大、小有腔卵泡中的各类细胞均有TSP-1和CD36蛋白的表达；在绵羊卵巢GCs中能明显检测到TSP-1和CD36阳性信号，且定位于颗粒细胞的细胞质中。（3）FSH通过与绵羊GCs上的FSHR结合，激活PI3K/Akt信号通路, 抑制了转录因子EGR1表达，EGR1与TSP-1基因启动子核心区域应答EGR1原件结合，抑制了TSP-1的表达。（4）FSH和siTSP-13可促进绵羊GCs的增殖, ABT-510可促进GCs凋亡，抑制GCs的活力；ABT510抑制了GCs中FSH诱导的孕酮和雌激素的合成；TSP-1通过影响组蛋白修饰促进StAR和HSD3B基因mRNA的表达，但在不同类固醇合成酶基因中的参与方式不同。（5）TSP-1模拟肽ABT510抑制了FSH处理后卵泡由中等大小卵泡向大卵泡发育的潜能，抑制了FSH对E2（雌激素），P（孕激素）的促进作用，而促进了FSH对LH的抑制作用，同时，ABT-510抑制了FSH对ER-α、ER-β和FSHR mRNA表达量的促进作用。FSH对CD36和VEGF的抑制或促进作用是通过抑制TSP-1实现的，提示TSP-1和CD36可能通过抑制血管生成影响卵泡的生长。以上研究内容为阐明TSP-1和CD36基因参与卵泡发育和颗粒细胞增殖和分化的分子机理提供理论和实验依据。