基于Wnt信号通路研究温肾活血中药干预对胚胎着床障碍小鼠的影响

Embryo implantation dysfunction is the key factor of low pregnancy rates in IVF-ET.The activate of embryo 、the endometrial receptivity and the inter-action of embryo and endometrial play great roles in the procedure of implantation. the process involves many signal pathway in the co-operation of endometrila/embryo , Wnt/β catenin signal pathway is identified and most important in the activationof embryos, decidualisation and the the implantation ,which may be regulated by both steroid hormone.Based on the theory of Traditional Chinese Medicine "palace cold infertility", we used "WENSHENHUOXUE "compound to treat repeatedly transplant failure patients, can significantly increase pregnancy rate, the mechanism may involves with improve progesterone levels and improve the endometrial receptivity. To further explore thepotential relationship between the mechanism of the "WENSHENHUOXUE"receip and the Wnt signal pathway .the present study will be investigated. To establish the implantation dysfunction mouse model with Mifepristone ,we set up groups as TCM A,TCM B(as high and low dose group of traditional Chinese medicine) and progesterone group,set model group 、normal pregant group and false pregnancy group as controlled groups. Get samples of embryo and endometrial at the day 2、4、6、8 of pregant.and to observe the expression of many Wnt related genes,protein and inhibitor of signaling pathway DKK1、FrpHE by immune fluorescence technology, immunohistochemical, RT-PCR, westernblot .to analyzes the relation of steroid hormone level and the signal pathway, which may provide scientific evidence to spread the use of the Chinese traditional medicine in IVF cycle.

胚胎着床障碍是IVF-ET低妊娠率的关键。种植窗期囊胚的激活及子宫内膜具有容受能力对于胚胎着床至关重要，涉及母/胚之间多种协同作用及信号传递，其中Wnt /βcatenin信号通路在胚胎激活、着床及内膜蜕膜化过程中均发挥重要作用，并受雌孕激素调控。基于中医"宫寒不孕"理论，既往采用"温肾活血复方"治疗反复移植失败患者能显著提高妊娠率，其机制与提高孕酮水平，改善内膜容受性相关。为进一步探讨该方对种植窗期囊胚与子宫内膜之间信号传递的影响，提出本课题研究。以米非司酮建立着床障碍模型设立中药组及黄体酮组，与模型组、正常组及假孕组小鼠对照，在妊娠D2、4、6、8天提取胚胎及子宫内膜，采用免疫荧光技术、免疫组化、RT-PCR、蛋白印迹等多种技术观察囊胚及内膜上Wnt信号通路相关基因、蛋白及抑制因子FrpHE和DKKl的表达，并分析雌孕激素水平与信号通路之间的相关性，为中医药介入IVF治疗提供科学依据。

胚泡着床障碍是导致IVF-ET失败的关键因素，在种植的过程中，囊胚与子宫内膜间的同步协调至关重要，Wnt(Wnt signaling pathway)信号通路被证实在促进囊胚的激活以及胚胎的着床方面发挥着重要的作用。基于中医“宫寒不孕”理论与既往研究证实“温肾活血方”能改善子宫内膜容受性从而提高妊娠率，本课题从胚胎与子宫内膜之间的相互作用出发，以米非司酮干预的胚胎着床障碍小鼠为模型，探讨“温肾活血方”对小鼠妊娠率和着床位点数、围着床期小鼠血清中雌孕激素及其受体水平、Wnt信号通路相关物质 mRNA及蛋白表达水平的影响。结果：一、温肾活血方可有效提高胚胎着床障碍小鼠的妊娠率及着床位点数。妊娠第8天，温肾活血方高、低剂量组妊娠率分别为90%(9/10)、90%(9/10)，明显高于模型组的妊娠率80%（8/10），且高、低剂量组的着床位点数13.78±2.774、10.33±1.323均显著高于模型组6.63±2.669，且P<0.01。二、温肾活血方能够有效改善着床障碍小鼠的子宫内膜组织学形态。其中，温肾活血方高剂量组宫腔上皮厚度（14.18±2.63）μm模型高于模型组（11.92±0.95）μm。三、温肾活血方能够改增加雌、孕激素受体浓度。其中妊娠D5天，温肾活血方高、低剂量组的雌激素受体浓度分别为798.50±124.87、777.25±156.13，较模型组（754.00±87.73）均明显增加；四、上调Wnt/β-catenin经典信号通路中Wnt4、Wnt5a、β-catenin等的表达、抑制FrpHE、DKK1的表达，从而实现胚胎的激活与着床。其中观察妊娠D8天Wnt4mRNA、Wnt5amRNA表达可见，模型组分别为0.0091±0.0025、0.0056±0.0013，明显低于温肾活血方组高（0.0226±0.0030、0.0144±0.0026）、低（0.0174±0.0022、0.0117±0.0016）剂量组，且P<0.01。本课题以多个角度、多层次的探索温肾活血复方改善种植期子宫内膜容受性的机制，有助于进一步阐明中药的起效机制，为中医药辅助IVF提供新的思路及治疗策略，推扬IVF与中医药的进一步发展。