氨肽酶N通过VEGF/VEGFR通路改善子宫内膜容受性的机制研究

Implantation rate and cycle pregnancy rate of IVF-ET are not ideal. The reason may due to a negative effect on endometrial receptivity by controlled super-ovulation treatments during IVF-ET. Studies indicated that endometrial blood flow is an important factor on endometrial receptivity. In our previous research, we applied “Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)” analysis to explore the influence of different controlled super-ovulation treatments of IVF-ET patients from a global proteomic perspective, to search the potential biomarkers associated with endometrial receptivity and embryo implantation. Aminopeptidase N (ANPEP) is one of the proteins which demonstrated differential expression between different treatment groups and also closely associated with endometrial receptivity. Further studies suggested that ANPEP is mainly located in endometrial stromal cells and highly expressed during “implantation window”. Overexpression of ANPEP in stromal cells promote the proliferation of vascular endothelial cells. Studies showed ANPEP is an important regulator of neovascularization via vascular endothelial growth factor (VEGF) in tumor cells. While the function of this protein in endometrial receptivity is still unknown. We hypothesize that ANPEP protein may play an important role in the regulation of endometrial receptivity via VEGF-VEGFR pathway angiogenesis mechanism. By using human endometrial stromal cell and human microvascular endothelial cell in vitro culture as models, the influence of ANPEP expression level on endometrial receptivity markers expression in endometrial stromal cell will be detected using ANPEP RNAi, ANPEP overexpression and VEGF/VEGFR pathway inhibitors as approaches. Subsequently, the culture medium of endometrial stromal cell will be used to co-culture with microvascular endothelial cells to explore whether ANPEP regulate endometrial angiogenesis via VEGF-VEGFR pathway. Hence, the vascular endothelial cell cavity formation function, cell migration and cell proliferation will be detected. By those studies, the effect and mechanism of ANPEP in regulating endometrial receptivity will be stated. Eventually, this study provide clues for finding new markers of endometrium receptivity evaluation and improving embryo implantation rate and pregnancy rate in assisted reproductive technology.

目前IVF-ET的胚胎种植率及周期妊娠率尚不理想，可能是由于控制性促超排卵改变子宫内膜容受性对胚胎着床产生不利影响。研究表明子宫内膜血流丰富程度与容受性正相关。课题组前期对应用不同方案患者种植窗期子宫内膜组织进行蛋白质组及生物信息学分析，发现氨肽酶N（ANPEP）为调控子宫内膜容受性潜在靶蛋白，进一步研究表明其主要定位于间质细胞，种植窗期表达最强，且可促进血管内皮细胞增殖。ANPEP在肿瘤细胞中可调节VEGF，但在子宫内膜中作用报道较少。本课题以人子宫内膜间质细胞、微血管内皮细胞体外培养及共培养技术为基础，采用慢病毒载体对间质细胞ANPEP进行过表达或表达沉默及同时加入通路阻断剂等策略，通过检测内皮细胞血管生成功能，多方面探讨ANPEP通过VEGF/VEGFR通路促血管生成改善子宫内膜容受性的机制。本研究可为寻找评价子宫内膜容受性新标志物提供线索，为提高临床种植率及妊娠率提供新的治疗思路。

子宫内膜容受性是胚胎成功植入的必要条件之一，改善子宫内膜容受性是辅助生殖技术（ART）临床上提高体外受精—胚胎移植的种植率和妊娠率的关键。本课题组前期应用高通量定量蛋白质组学分析，发现在不同控制性促超排卵方案患者种植窗期的子宫内膜存在明显的蛋白差异化表达，氨肽酶N（ANPEP/APN/CD13）可能是参与调控子宫内膜容受性的潜在靶蛋白。研究发现各年龄段小鼠子宫内膜均表达ANPEP蛋白，在幼龄组最低，性成熟组最高；而后随着年龄的增长，ANPEP的表达随着生育能力的衰退而下降 (P<0.05)。ANPEP蛋白在人类生殖周期各阶段子宫内膜间质细胞中均表达，种植窗期组表达较增生期组及种植窗前期组明显增强(P<0.05)。子宫内膜间质细胞的增殖、迁移和侵袭能力随ANPEP过表达而增强，随其下调而减弱(P＜0.05)。随子宫内膜间质细胞的ANPEP表达上调，与之共培养的HUVECs的迁移和侵袭能力增加，反之下降（P<0.05）；用其培养液上清培养的HUVECs增殖活力、管腔形成能力随hESCs的ANPEP表达上调而增强，反之下降（P<0.05）。子宫内膜间质细胞的ANPEP表达上调，促进了子宫内膜容受性因子HOXA10、intergrinβ3、LIF的表达（P<0.05），而IL-1的升高趋势无统计学意义（P>0.05）；下调hESCs的ANPEP表达，HOXA10、intergrinβ3、LIF的表达下降（P<0.05），而IL-1无明显改变（P>0.05）。上调子宫内膜间质细胞的ANPEP表达，金属基质蛋白酶MMP-2、MMP-9的表达上调（P<0.05）；下调hESCs的ANPEP表达，MMP-2、MMP-9表达下降（P<0.05）。子宫内膜间质细胞的ANPEP表达可增加血管生成通路因子VEGF、VEGFR-2及PI3K、AKT的表达（P<0.05）；hESCs的ANPEP表达下调可引起VEGF、VEGFR-2及PI3K、AKT表达下降（P<0.05）。本研究结果提示子宫内膜间质细胞中ANPEP的表达可促进脐静脉内皮细胞成管能力，从而影响子宫内膜局部血管形成而参与调节内膜容受性，上调VEGF及其受体的表达是其发挥作用的机制之一。