中药黄芩、白术改善胚胎着床的糖分子机制研究

The development of the pregnancy-assisting technology such as fertilization - embryo transfer (IVF-ET) in vitro have made some breakthroughs in treatment of female infertility, however the pregnancy rate only reach 20% ~ 30%. The studies have shown that embryo implantation dysfunction is one of the main reasons for low pregnancy rate with IVF-ET. Traditional Chinese medicine in promoting embryo implantation have a good effect. The compatibility of Scutellaria baicalensis and Largehead Atractylodes Rhizome historically was called as one of the the tocolysis St. drugs. But so far few studies of the mechanism of facilitating pregnancy and soothing fetus with the Scutellaria baicalensis and Largehead Atractylodes Rhizome. The investigation is to establish human implantation model which can simulate the environment that in vivo. And the investigation will be performed on embryo implantation dysfunction model in vitro interfered by RNA and induced by mifepristone and embryo implantation dysfunction model in vivo in mice respectively. The change of implantation rate of and the oligosaccharides and its key enzyme of oligosaccharide synthesis will be analyzed by real-time PCR, Western blotting and immunohistochemistry before and after treatment of Scutellaria baicalensis and Largehead Atractylodes Rhizome. And the hypothesis is proposed that Scutellaria baicalensis and Largehead Atractylodes Rhizome can improve the embryo implantation by regulating the expressions of implantation-related oligosaccharides and the key enzyme of oligosaccharide synthesis. This project is related to the cross-cutting fields of the sugar medicine, Chinese medicine and assisting-reproductive technology. The theoretical foundation will be provided that Scutellaria baicalensis and Atractylodes are utilized in IVF-ET technology.

体外受精-胚胎移植(IVF-ET)等现代助孕技术的发展使女性不孕症的治疗取得了突破性进展，但妊娠率只能达到20%～30%。研究表明，胚胎着床障碍是导致IVF-ET妊娠率低的主要原因之一。传统中药在促进胚胎着床方面有很好的疗效。黄芩、白术配伍历史上誉为"安胎圣药"。 但迄今有关黄芩、白术促孕安胎的机制研究较少。本课题建立了能模拟在体着床特性的人体外胚胎着床模型，并经RNA干扰和米非司酮分别诱导后建立离体胚胎着床障碍模型和小鼠在体胚胎着床障碍模型进行研究。通过Real-time PCR、Western杂交、免疫组织化学等细胞分子生物学技术分析了黄芩、白术治疗后着床率的变化及着床相关寡糖和寡糖合成关键酶的变化，提出黄芩、白术通过调节着床相关寡糖和寡糖合成关键酶的表达来改善胚胎着床的见解。此课题是糖理论研究与中药、辅助生殖技术相结合的交叉领域。为黄芩、白术在IVF-ET 技术中的应用提供理论依据。

体外受精-胚胎移植(IVF-ET)等现代助孕技术的发展使女性不孕症的治疗取得了突破性进展，但妊娠率只能达到20%～30%。研究表明，胚胎着床障碍是导致IVF-ET妊娠率低的主要原因之一。传统中药在促进胚胎着床方面有很好的疗效。黄芩、白术配伍历史上誉为“安胎圣药”。 但迄今有关黄芩、白术促孕安胎的机制研究较少。 本研究成功建立了能够模拟在体着床特性的RL95-2和JAR人体外胚胎着床模型系统，并经米非司酮诱导和FUT4RNA干扰后建立离体胚胎着床障碍模型和小鼠在体胚胎着床障碍模型进行研究。经黄芩、白术水煎液治疗后观察胚胎着床率的变化，通过RT-PCR、Western blot等生物学技术分析了黄芩、白术治疗障碍模型前后，着床相关寡糖LeY、sLeX和寡糖合成关键酶FUT4、7的表达变化。. 离体实验RT-PCR从基因水平分析显示中药黄芩、 白术治疗后FUT4、7表达明显高于障碍模型组。Western blot检测中药治疗后着床相关寡糖LeY、sLeX和寡糖合成关键酶FUT4、7的表达也明显高于障碍组。在黄芩、白术对胚胎粘附率的影响实验中，对照组JAR细胞与RL95-2细胞的黏附率为69.5％，障碍模型组黏附率是22.4 %其黏附率较对照组明显减少，中药治疗障碍组后细胞的黏附率为35.4％较障碍组明显升高。组间差异具有统计学意义(*p<0.05)。. 在体实验孕5天时处死小鼠取子宫内膜做RT-PCR实验。从基因水平分析显示中药黄芩、 白术治疗后FUT4、7表达明显高于障碍模型组。Western blot检测中药治疗后着床相关寡糖LeY、sLeX和寡糖合成关键酶FUT4、7的表达也明显高于障碍组。孕8天时处死小鼠剖腹 , 取出子宫检查胚胎着床数 ,对照组平均胚胎着床数23.32 ± 3.68，障碍模型组平均胚胎着床数3 .00 ± 2.00，较对照组明显减少。中药治疗组平均胚胎着床数15.6 ± 3.603较障碍组明显升高。组间差异具有统计学意义(*p<0.05**P<0. 01)。. 本研究是糖理论研究与中药应用、生殖医学相结合的交叉领域。以现代医学方法从分子生物学水平证实中药黄芩、 白术在胚胎着床中的促进作用,以及中药黄芩、 白术可通过调节着床相关寡糖sLeX、LeY以及寡糖合成关键梅FU4、7的表达来促进胚胎着床的见解。