EGF相关生长因子在人卵巢颗粒细胞VEGF生成中的作用及分子调控机制

Aamphiregulin, betacellulin and epiregulin belong to the epidermal growth factor(EGF)family. These three EGF-like growth factors can be induced by LH surge rapidly and transiently in human granulosa cells and they play significant role in regulation of ovarian function as LH mediators. Vascular endothelial growth factor(VEGF), which is mainly produced by human granulosa cells, is also up-regulated by LH surge. In normal female reproductive physiology, VEGF is an important factor in the follicular development and luteinization, however, the over-expressed VEGF may cause ovarian hyperstimulation syndrome(OHSS)during IVF process. Our preliminary results showed that these EGF-like ligands can up-regulate VEGF mRNA expression in human granulose cells, however, the underlying molecular mechanism is still unknown.It is proposed that these three EGF-like ligands can up-regulate VEGF production by binding to ErbB family,phosphorating ERK1/2 or Akt signaling pathway,then activating specific transcript factor. In the current study, our first aim is to examine the effects of EGF-like ligands on VEGF expression in human granulosa cells and the underlying molecular mechanism.By using inhibitor or siRNA technics, our second aim is to find the method to block the production of VEGF. These results provide not only important insights into the molecular mechanisms mediating EGF-like growth factor-regulated OHSS but also targets that can be used for the prevention and treatment of OHSS.

双调蛋白、β细胞素和表皮调节素属于表皮生长因子家族(EGF)，LH峰后其在人颗粒细胞内表达升高，是LH发挥作用的重要媒介物质。人颗粒细胞来源的血管内皮生长因子(VEGF)也在LH峰之后表达升高，生理情况下参与卵泡发育和黄素化过程，而在辅助生殖助孕过程中注射HCG之后，VEGF过表达则引起卵巢过度刺激综合症(OHSS)。我们前期研究已证实上述三个生长因子上调VEGFmRNA的合成,推测其作用机制可能为通过与EGF受体家族不同受体结合，激活胞内ERK1/2或Akt信号通路，活化特异性转录因子促进VEGF的合成。本课题将在受体水平、胞内信号转导通路水平及核内转录因子水平上分别采用特异性抑制剂或RNA干扰方法阻断VEGF的合成，研究上述三个生长因子促进VEGF合成的分子机制，寻找阻断VEGF合成的方法，为预防和治疗OHSS提供新的思路和方法。

EGF相关因子包含双调蛋白（Amphiregulin，AREG）、β细胞素（Betacellulin，BTC）和表皮调节素（Epiregulin，EREG），是LH发挥作用的重要媒介物质，人颗粒细胞来源的血管内皮生长因子（Vascular endothelial growth factor，VEGF）也在LH峰之后表达升高，生理情况下参与卵泡发育和黄素化过程，而在辅助生殖助孕过程中注射hCG之后，VEGF过表达则引起卵巢过度刺激综合症(Ovarian hyperstimulation syndrome，OHSS)，OHSS 是辅助生殖技术助孕过程中最严重的并发症，目前尚无有效早期预防和治疗方法。本课题发现AREG、BTC和 EREG在人卵巢颗粒细胞促进VEGF mRNA表达和蛋白生成，三者之间无协同作用，其中具有关键作用的生长因子是AREG；发现AREG、BTC和 EREG在人卵巢颗粒细胞中通过两条通路促进VEGF生成，其一通过COX-2-PGE2间接通路，另一条通过EGFR-ERK直接信号通路；另一方面，本课题首次报道OHSS患者卵泡液AREG蛋白浓度及颗粒细胞AREG mRNA表达量高于正常对照组，卵泡液内AREG蛋白含量与窦卵泡数、获得卵子数，hCG日血清内雌二醇水平、取卵日血清雌二醇水平、取卵后48小时血清雌二醇水平均呈正相关关系；OHSS患者壁颗粒细胞表面EGFR及HER2 mRNA的表达水平明显高于正常对照组；OHSS患者卵泡液及颗粒细胞VEGF高表达，卵泡液AREG浓度与VEGF浓度呈正相关；AREG在OHSS患者颗粒细胞中能更快更大幅度上调VEGF表达；hCG可能通过AREG-EGFR介导VEGF生成。本课题从全新的角度解释OHSS发病机制，为预防和治疗OHSS提供新思路。