利用体外血管生成模型研究TGFβ1/Smads信号通路调控牛黄体血管生成的分子机制

Corpus luteum (CL) formation is accompanied by extensive angiogenesis, this process is essential for sufficient progesterone to be produced to support and maintain of early pregnancy. Therefore, the angiogenic process in the developing CL is critical for its function. Recently, we used a novel luteinising follicular culture system that mimics luteal angiogenesis and found that transforming growth factor β1 (TGFβ1) inhibited the angiogenesis, participated in the disassembly of the microvasculature during follicle-luteal transition, but the underlie molecular mechanisms were unknown. Various studies have confirmed that TGFβ1 plays its role through TGFβR/Smads signaling pathway. Thus, we speculate that TGFβ1 bind to its receptors (ALK5) and then activate Smad-2 and Smad-3, thus blocks angiogenesis in CL by inhibiting endothelial cells proliferation. To confirm this, this project will mainly conduct on the follicular culture system treated with TGFβ1, expression of ALK and phosphorylation level of Smads expression will be assayed. Furthermore, the receptor inhibitor SB505124 and K02288 will used to block the signal pathway, and changes in phosphorylation Smads expression and ALK expression will also be investigated. Taking all these together, this project will identify and unravel the underlying molecular and cellular mechanisms of the effect of TGFβ1 on angiogenesis in CL, confirm that the regulation effect of TGFβ1 on the luteal angiogenesis through TGFβR/Smads. In addition, the expression of key enzymes during progesterone secretion will be detected to determine whether TGFβ1 inhibit the steroidogenesis capacity of luteal cells. This work will contribute to the regulatory mechanism of luteal angiogenesis, and hope it could provide theoretical foundations for developing of novel strategies to alleviate the luteal inadequacy infertility and improve the animal fertility.

黄体（CL）中的血管网络确保合成充足的孕酮以建立和维持妊娠，对于其功能发挥至关重要。申请人前期利用牛卵泡细胞培养体系模拟CL中血管生成的研究发现，转化生长因子（TGFβ1）抑制血管生成以及孕酮分泌，但具体的分子机制亟待确定。TGFβ1通过TβR受体/Smads信号通路发挥作用，我们推测：TGFβ1与受体ALK5结合后激活Smad-2/-3，抑制了血管内皮细胞增殖。本研究拟在模拟CL血管生成过程时添加TGFβ1后ALK表达和Smads磷酸化水平为主线；以受体抑制剂SB505124和K02288对Smads信号通路影响为桥梁；揭示TGFβ1调控调控血管生成是通过TβR/Smads信号通路介导的。此外，检测孕酮合成关键酶（StAR、P450scc、3β-HSD等）的变化确定TGFβ1是否抑制CL的分泌功能。研究结果将补充CL血管生成的复杂调控机制，有助于改善动物繁殖性能以及黄体不足不孕症的治疗。

黄体中的血管网络确保合成充足的孕酮以建立和维持妊娠，对于其功能发挥至关重要。卵泡-黄体转化过程中的血管生成受到大量因子的复杂调控，如包括血管内皮生长因子、转化生长因子β（TGF-β1）等，而TGF-β1调节早期黄体中的血管形成的作用机制并不明确。本项目利用牛卵泡细胞体外培养系统模拟卵泡-黄体转化过程中血管形成过程时添加TGF-β1，检测对下游信号分子的影响；并利用TGF-β1受体的特异抑制剂SB431542阻断TGF-β1信号通路传导后对黄体血管生成的影响为桥梁，再结合RNA-seq分析确定起关键作用的血管生成相关基因。研究结果发现：TGF-β1通过其上游分子LTBP活化受体TβRI，随后磷酸化Smad2、Smad3、ERK1/2以及PI3K/Akt蛋白，抑制了eNOS的表达，上调了血管生成相关的SERPINE1、SERPINB5的表达，通过抑制CLDN1、SDC1、ESAM的表达阻止了细胞粘附连接，但是上调了细胞基质受体 SDC1的表达；而且通过抑制AREG、EREG、HB-EGF的表达抑制了ErbB2信号通路的激活；此外TGF-β1还通过下调CYP11A1和3β-HSD的表达来抑制孕酮的分泌，但是对细胞的增殖和凋亡均没有明显影响，证明了TGF-β1对黄体细胞中孕酮分泌的抑制作用是由类固醇分泌功能（孕酮合成关键酶）被抑制造成的。本项目的研究阐明了TGF-β1对早期黄体中血管生成的调控作用是通过TβR/Smads信号通路介导细胞粘附连接的破坏以及细胞基质降解的阻止造成的，而ERK1/2、ErbB2和PI3K信号通路在该过程中起到了间接调节作用。该项目建立了TGFβ-1与黄体血管生成被抑制两者之间的细胞内信号调节机制。丰富和发展了黄体中血管生成的复杂调控机制的研究，有助于开发新的技术用于治疗黄体不足以及改善动物繁殖性能。