人白细胞抗原G在滋养细胞侵润迁移中的功能和相关机制研究

In this context it becomes critically important to understand, at a molecular level, placental development, which determines the organ’s functional capacity. This paper focuses on one part of this process: trophoblast differentiation, a component that is integral to implantation and trophoblast invasion of the uterus. The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. In our previous work, we found novel roles for HLA-G in regulating b-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion. So we infer that HLA-G may also play a role in regulating trophoblast cell invasion and migration. The goal of our proposed research is to indicate the role of HLA-G playing in trophoblast invasion and migration, and its mechanism. To achieve our goals, we will perform the following research: 1) trophoblast invasion and migration through transwell insert, 2) study the signal transduction pathway HLA-G involved, 3) examine the actin stress fibers and focal adhesions in HLA-G expressing cells, 4) explore the HLA-G interacting molecule combining with immunoprecipitation and LC-MC/MC assay. Accomplishing our proposed research will provide direct evidences that HLA-G may be a possible biomarker of embryonic developmental potential to predict human-assisted reproduction outcomes.

人类胚胎着床和胎盘种植的相关机制是生殖医学研究的重点问题。以往的研究显示，人白细胞抗原G（HLA-G）在胎盘滋养细胞的表达缺失与习惯性流产密切相关；同时在辅助生殖技术中，早期胚胎表达HLA-G与妊娠结局成正相关。但是HLA-G影响滋养细胞对母体子宫的适度侵润的机制目前尚不明确。本项目采用慢病毒介导的稳定基因重组技术和Transwell迁移模型研究HLA-G对滋养细胞侵润迁移能力的影响；采用基因组学技术，研究HLA-G对滋养细胞侵润迁移相关因子和基因表达谱的影响；采用磷酸蛋白质组学技术研究HLA-G影响的信号转导途径；采用质谱分析和免疫共沉淀技术，研究滋养细胞中与HLA-G相互作用的蛋白分子。通过上述研究，了解HLA-G对滋养细胞侵润迁移的影响和相关机制，揭示HLA-G在人类胎盘植入中的作用。本研究对于进一步阐明人类早期胚胎着床的分子机制具有重要的理论意义。

滋养细胞的分化和功能维持对胚胎植入和发育，胎盘形成和其对母体的侵润起至关重要的作用，因此在分子水平研究胎盘发育和滋养细胞分化对深入理解妊娠维持和妊娠相关疾病发病机制极为重要。人白细胞抗原G（HLA-G）是滋养细胞特异表达的非经典HLA I类分子，临床资料显示，HLA-G表达水平下降影响辅助生殖的妊娠结局，并与习惯性流产、先兆子痫等病理妊娠的发生密切相关。.滋养细胞对母体子宫的适度侵润与迁移是保证成功妊娠的重要事件。通过适度地侵润和迁移，滋养细胞侵润母体子宫内膜和肌层的上三分之一从而将胎盘和胎儿锚定到母体子宫，同时滋养细胞还可以替换母体螺旋动脉内皮细胞，完成母体血管重建，保证胎盘的血液供应。HLA-G表达于侵润子宫内膜和内1/3肌层的绒毛外滋养细胞和重塑螺旋动脉的血管内滋养细胞，在胎儿-母体界面发挥免疫调控作用，保护胎儿来源的滋养细胞在侵润子宫壁时免受母体免疫系统攻击。为研究HLA-G对滋养细胞迁移的影响和调控机制，采用慢病毒感染，建立稳定表达HLA-G的人绒毛外滋养细胞HTR8/SVeno细胞系和人绒毛膜癌BeWo细胞系。通过Transwell cell migration assay检测了HLA-G对滋养细胞迁移能力的影响，发现HLA-G可调控滋养细胞迁移，表达HLA-G的滋养细胞迁移能力显著升高，敲低HLA-G可抑制细胞迁移。在表达HLA-G的滋养细胞中Rac活性上调，RhoA活性降低以促进黏着斑的降解，抑制肌动蛋白张力纤维的合成，从而引起细胞迁移能力的增加。另外，抑制ROCK活性可补偿不表达HLA-G的滋养细胞迁移能力。上述结果显示HLA-G作用于ROCK的上游以影响Rho通路的活性促进滋养细胞迁移。HLA-G 的表达可上调滋养细胞细胞内调控细胞骨架的上游分子H-Ras的活性而不影响滋养细胞增殖。利用质谱技术，首次鉴定出HLA-G 可与 RIN1 分子特异性结合促进滋养细胞迁移。HLA-G 与 RIN1 结合后，促进 c-ABL 磷酸化并活化 Ras，激活Ras 下游的 PI3K 通路活化 Rac1/2/3，同时抑制 RhoA/C 活性，最终影响胞内黏着斑和细胞骨架的重组，促进滋养细胞迁移。