从血管形成与黄体构建研究补肾、活血改善COH着床障碍及早期妊娠丢失的机制

In view of the inadepuate luteal function in controlled ovarian hyperstimulation (COH) and the importance of Corpus luteum (CL) function in embryo implantation and pregnancy maintenance and the effect of angiogenesis on CL morphology and function, combined with the clinical fact that the methods of Bushen and Huoxue are applied during the treatment processes of regulating menstrual cycle and enhancing ability to conceive as well as preventing abortion, some hypotheses are suggested as follows based on the previous work: (a) the component of Huoxue not only can faciliate the development and maintenance of the CL, but also promote the invasion of trophoblast cells on the maternal-fetal interface, probably by promoting angiogenesis; (b) the component of Bushen can coordinate hormone levels and expression of related factors at the implantation site among ovary, CL, and maternal-fetal interface; (c) The components of Bu-Shen-An-Tai recipe (BSATR) function synergistically, and the whole BSATR makes the synchronous and well-organized development between ovary and CL as well as maternal-fetal interface, which is conducive to improving the success rate of gestation. In this study, the mouse COH model serve as the research object. The methods of molecular biology, morphology and electron microscopy, as well as dynamic or continuous sampling methods were applied to investigate the success rate of gestation, levels of hormones and related factors as well as their receptors such as LH, hCG, P4, VEGF, FGF, PGF2, PGE2, PGES and PGFS in the serum, ovary, and endometrium, and also ultrastructure of CL and endometrium in the mouse COH model. This study will provide experimental foundation for the therapy of infertility by combining the principle of Bushen and Huoxue with modern reproductive medicine, especially the effect of Huoxue on angiogenesis in the infertility treatment.

鉴于COH中存在黄体功能不足和黄体功能在胚胎着床及早期妊娠中的重要地位，以及血管形成对黄体形态和功能的影响，结合补肾、活血贯穿于调经种子安胎治疗全过程的临床事实，在前期工作基础上，提出假说：活血组分可能通过促进新生血管形成，既有利于黄体形成与维持，也可促进母胎界面滋养细胞侵入；补肾组分可协调卵巢、黄体、母胎之间激素及相关因子的表达；两组分相互协作，有助于卵巢、黄体、母胎界面之间同步、有序地发育，改善着床障碍，防止胚胎丢失，提高孕育成功率。将以控制性超排卵小鼠模型为研究对象，运用分子生物学、免疫组化、形态学定量以及电镜等技术，采取动态、连续取样的方式，对模型小鼠的孕育成功率，血清、卵巢、子宫内膜中LH、hCG、P4、VEGF、FGF、PGF2、PGE2、PGES、PGFS及其受体含量，黄体、子宫内膜超微结构进行观察，证明假说，为补肾、尤其是活血法则与现代生殖医学相结合治疗不孕症提供实验依据。

不孕症是常见病，辅助生殖技术在临床中越来越多地被用于不孕症的治疗。但是，控制性超促排卵作为体外受精——胚胎移植的重要环节，可能导致胚胎着床率和活产率低下。鉴于中医的补肾、活血贯穿调经种子安胎治疗全过程的临床事实，本课题以控制性超促排卵小鼠模型为研究对象，深入研究了补肾安胎方改善COH小鼠胚胎着床障碍的作用机制。研究结果表明，控制性超促排卵显著降低了胚胎着床率和子代小鼠活产率，且导致子代雌性小鼠的生殖内分泌功能紊乱；补肾安胎方（补肾活血方）低、中、高各剂量及补肾、活血组分能够不同程度地改善COH小鼠胚胎着床率，其作用机制如下：1）该方减低卵巢过激，减少卵巢颗粒细胞的凋亡，改善黄体功能；2）该方能有效调节模型小鼠整体雌、孕激素水平，改善小鼠子宫内膜雌、孕激素受体及其相关作用通路（ER/LIF/HB-EGF）的表达；3）该方能增加子宫内膜胞饮突及子宫内膜容受性相关分子（E-cadherin、Integrin β3、PCNA、Hoxa 10、Ihh）的表达；4）该方能促进围着床期子宫内膜的组织重塑与血管形成。本课题从多个环节、多个层面揭示了中医补肾、活血在胚胎着床环节的作用机制，为补肾、尤其是活血法则与现代生殖医学结合治疗不孕症提供了实验依据。