Emmprin和TGF-β调控子宫内膜MMPs表达的信号转导机制及其与奶牛不孕症关系的研究

The imbalance of matrix metalloproteinases (MMPs) which impedes extracellular matrix's degradation and embryo nidation is one of the major causes of infertility. The expression of MMPs is in the two-way control of Emmprin and TGF-β which are identified as MMPs' inducing factor and inhibiting factor. But the regulatory mechanism is still unclear, so is the mechanism of infertility caused by the imbalance of MMPs. TGF-β signal transduction is centred around Smad. Our research confirmed that the acceleration effect of Emmprin on the expression of MMPs is directly related to mitogen activated protein kinase (MAPK) p38's phosphorylation. Many methods are intended to carried out in this research, such as RNA interference, gene overexpresstion, laser scanning confocal microscope, Western blot, real-time PCR array and so on to study the influence of Emmprin on MAPK pathway's, TGF-β1 on Smad pathway's gene expression profile, and the integration of the Co-localization of Caveolin-1, Emmprin and TβRI on related signaling pathways. This research will focus on studying the characters of the MMPs' key regulatory molecules in infertile cows endometrium and the isostatic compensation of signaling pathways regulation on the MMPs. Furthermore, it will indicate the signaling mechanism of Emmprin and TGF-β's regulation on endometrium MMPs and the relationship of them with the cow's infertility, providing scientific basis for the preventive and therapeutic strategies of infertility.

基质金属蛋白酶(MMPs)失衡使细胞外基质降解受阻，胚胎着床障碍，是引起不孕的主要原因之一。MMPs表达受其诱导因子Emmprin和抑制因子TGF-β双向调控，但调控机制尚不明确，使MMPs失衡致不孕的机理仍不清楚。TGF-β信号转导以Smad为中心，我们研究证实 Emmprin促进MMPs表达与磷酸化MAPK p38直接相关。为此本项目拟采用RNAi、基因超表达、共聚焦显微镜、Western blot、实时定量PCR芯片等方法，研究子宫内膜上皮细胞Emmprin对MAPK、TGF-β对Smad信号通路基因表达谱的影响，小窝蛋白-1与Emmprin、TβRI共定位对调控MMPs信号通路的整合作用；分析不孕奶牛子宫内膜MMPs及其关键调控分子表达特征，信号通路调节对MMPs的均衡作用，揭示Emmprin、TGF-β调控子宫内膜MMPs的信号转导机制及与不孕的关系，为奶牛不孕防治提供科学依据。

基质金属蛋白酶（MMPs）通过降解细胞外基质（ECM）调控子宫内膜微环境，是导致子宫内膜容受性降低的关键因素，与奶牛不孕症密切相关，深入研究MMPs表达调控机制，有助于揭示奶牛不孕症的发病机理。结果显示，屡配不孕病牛子宫内膜胶原蛋白、层粘连蛋白、纤粘连蛋白显著高表达(P<0.05)，MMP-1、-2、-3、-9 mRNA显著低表达(P<0.05)，说明MMPs降解ECM能力下降，ECM高表达阻碍胚胎着床；通过RNA干扰降低子宫内膜上皮细胞Emmprin mRNA表达可致MMPs mRNA表达量显著下降(P<0.05)，同时7个p38MAPK通路蛋白、4个细胞周期蛋白差异表达下调，且MAPK抑制剂中仅p38MAPK抑制剂可使子宫内膜上皮细胞MMPs mRNA表达显著下调，表明p38MAPK通路为上调MMPs表达的主要路径；经TGF-β1(5ng/mL)处理子宫内膜上皮细胞，可显著提高Smad2、3的磷酸化水平，TRI、Smad2、Smad3、CoL3A1、CoL1A、PAI-1等与ECM代谢相关的基因均差异表达上调，说明TGF-b致纤维化作用是通过Smad途径实现的；通过增强Caveolin-1表达和降低Emmprin表达，可见细胞膜Emmprin的减少可促进Caveolin-1与TbRⅠ高效结合，证实Caveolin-1通过与Emmprin和TbRⅠ之间平衡，来调控MMPs表达；健康奶牛发情期和发情后期p38MAPK、MMPs高表达，Smad2、3低表达，而屡配不孕奶牛子宫内膜p38MAPK、MMPs低表达、Smad2、3高表达，表明调控MMPs表达的信号通路是引起子宫内膜ECM失衡的关键因素。本研究揭示了Emmprin/MAPKp38信号通路为上调奶牛子宫内膜MMPs表达主要调节路径，而TGF-β/Smad信号通路为抑制MMPs表达的主要路径，Caveolin-1在细胞膜水平通过与Emmprin和TβRI的选择性结合而对MMPs表达的两个通路起到整合作用，屡配不孕奶牛子宫内膜容受性降低与p38MAPK、Smad通路关键蛋白的异常表达致MMPs失衡密切相关。