控制性超促排卵对种植窗期uNK细胞及其调控下的血管生成通路时空表达与分布的影响及补肾法的干预

Poor endometrial receptivity coursed controlled ovarian hyperstimulation(COH) is an important factor of embryo implantation failure. It is known that angiogenesis is key step in embryo successful implantation. Research showed that uterine natural killer cell(uNK) is closly related with angiogenesis in implantation window. This research early clinical observation showed that Kindey-reinforcing therapy can regulate endometrial receptivity and improve the clinical pregnancy rate in ovarian stimulation cycle. We speculate that poor endometrial receptivity coursed COH is related to uNK cells and angiogenesis deficiency under the regulating of uNK cells, which is the internal relevant with the mechanism of Chinese medicine kinder-deficiency coursing uterus blood- deficiency is difficult to keep subtle materials inside uterus. In order to confirm the hypothesis, the study designed a series of experiments. This research includes both in vivo and vitro experiments. Observe the Kinder-reinforcing therapy effect on the expressions of time and space, distribution change of uNK cells and angiogenesis under the regulating of uNK cells in COH cycle before implantation, after implantation, implantation spot, non-implatation spot. Analyze the effects of Kinder-reinforcing therapy mediated uNK cells regulating angiogenic factors expression. In order to clear Kinder-reinforcing therapy have the effects of promoting endometrial receptivity from the point view of angiogenesis micro-environment of embryo implantation .At mean time, it is helpful for further understand the connotation of Chinese medicine keeping subtle materials inside uterus, also can be provide a new research mode for Chinese medicine prevention and treatment embryo implantation failure.

控制性超促排卵(COH)造成子宫内膜容受性受损是胚胎种植失败的重要因素。已知血管生成是胚胎着床成功的关键环节，研究表明子宫内膜自然杀伤（uNK）细胞与种植窗期血管生成密切相关。本课题前期临床研究证实，补肾法可改善促排卵周期子宫内膜容受性。我们推测COH干预后内膜容受性受损与胚胎种植微环境中uNK细胞及其调控下的血管生成障碍有关，这与中医“肾虚胞宫精血乏源难以摄精容物”的病机有着内在联系。为证实这一假说， 本研究拟通过动物实验结合细胞实验，观察补肾法对COH周期种植前后、着床点与非着床点uNK细胞及调控下的血管生成相关因子时空表达、分布变化的影响，分析补肾法介导人子宫内膜uNK细胞调控血管生成因子表达的作用。以期从胚胎着床血管生成微环境入手，明确补肾法在促进COH后子宫内膜向容受态转化中的作用。有助于进一步理解中医胞宫“摄精容物”的内涵，也能为中药防治胚胎着床障碍性疾病提供新的研究模式。

目前控制性超排卵（COH）造成子宫内膜容受性受损已成为影响辅助生殖技术（ART）妊娠率的重要因素。因此，改善COH后种植窗期胚胎移植的微环境、提高子宫内膜容受性，突破IVF低妊娠率的瓶颈，是ART面临的现实难题，也是研究者们密切关注及倾力研究的关键点。本研究以uNK细胞及其调控下的血管生成相关通路为核心，观察补肾法对种植前后、着床点子宫内膜形态、uNK细胞以及血管生成相关因子时空表达的影响，从子宫内膜微环境探索补肾法改善子宫内膜容受性的机制。因此，本项目主要研究包括（1）观察 COH 对种植窗口期子宫内膜血管生成微环境的影响；（2）检测补肾法改善COH干预后种植窗口期子宫内膜血管生成微环境的分子途径。通过注射GnRH-α+HMG+HCG，成功建立小鼠超促排卵模型，将模型组分为4组，另外3组分别添加寿胎丸高、中、低剂量干预，另设1空白组进行对照。连续观察着床窗口期子宫内膜形态、血管生成相关因子及胞饮突的变化。结果显示：（1）COH作用后着床窗口期子宫内膜发育不良，电镜下胞饮突少见。内膜局部CD56+标记的uNK细胞及其血管生成相关因子TGF-β1、MVD、Ang1、Tie2、VEGF、GM-CSF表达下调，表明COH可影响子宫内膜血管生成的微环境，降低子宫内膜容受性；（2）补肾中药干预后，内膜腺体及间质均得到明显改善，胞饮突发育完全，且数量增多。uNK细胞及其血管生成相关因子表达上调，并且随着妊娠进展表达逐渐升高。表明补肾法在促进COH干预后子宫内膜向容受状态转化中具有积极的作用。促进血管生成相关因子的表达是其改善子宫内膜容受性的途径之一。这为中医药干预子宫内膜容受性调节的新靶点提供依据，也有助于进一步理解中医胞宫“摄精容物”的内涵，对中医药补肾促血管生成防治胚胎着床障碍（胚胎反复移植失败，复发性流产）、不明原因不孕症等妊娠疾患具有重要意义。