补肾安胎冲剂改善RSA小鼠母胎界面血管生成障碍的机制研究

The obstacle of vascular remodeling in Maternal-fetal interface is one of the important reasons of recurrent spontaneous abortion (RSA). Bushenantai Granules, which to kidney and spleen tocolysis for treatment, have a clear improvement in the maternal-fetal interface angiogenesis. The subject is on the basis of previous studies, establishing RSA mouse models. It uses qRT-PCR, Western blot and other methods to detect protein and gene expression of VEGF and VEGFR-2, sFlt – 1,HIF-1, , TGF - β, VHL, E2 in the serum and decidua, which can observe the expression of VEGF, Ras, MAPK, PI3K, AKT in placental microvascular endothelial cells, smooth muscle cells and The obstacle of vascular remodeling in Maternal-fetal interface is one of the important reasons of recurrent spontaneous abortion (RSA). Bushenantai Granules, which to kidney and spleen tocolysis for treatment, have a clear improvement in the maternal-fetal interface angiogenesis. The subject is on the basis of previous studies, establishing RSA mouse models. It uses qRT-PCR, Western blot and other methods to detect protein and gene expression of VEGF and VEGFR-2, sFlt – 1,HIF-1, , TGF - β, VHL, E2 in the serum and decidua, which can observe the expression of VEGF, Ras, MAPK, PI3K, AKT in placental microvascular endothelial cells, smooth muscle cells and fibroblasts to explore Bushenantai granules on the regulation of angiogenesis network. It takes human placental microvascular endothelial cells as the research object under the condition of three-dimensional cultivation, using gene chip technology, RNAi and overexpression techniques, sequential signal blocking and other means to explore the influence of traditional Chinese medicine on Ras/MAPK and PI3K/AKT signaling pathway cross-talk which VEGFR-2 mediates and to clarify the mechanism of molecular biology of improving the angiogenesis obstacle in Maternal-fetal interface in RSA mice by using kidney and spleen traditional Chinese medicine ,provide ideas and methods for clinical treatment as well as enrich traditional Chinese medicine “cellular vein systems in the kidney” the scientific connotation.

母胎界面血管生成障碍是复发性流产的重要因素之一。补肾安胎冲剂以补肾健脾立法，有明确的改善母胎界面血管生成的作用。本课题在前期研究的基础上，建立RSA小鼠模型，用qRT-PCR、Western blot等方法，检测血清、蜕膜VEGF、VEGFR-2及sFlt-1、HIF-1、TGF-β、VHL、E2等调控因子的表达，观察VEGF、Ras、MAPK、PI3K、AKT在胎盘微血管内皮细胞、平滑肌细胞及成纤维细胞的分布，探索补肾安胎冲剂对血管生成网络的调控；以三维培养条件下的人胎盘微血管内皮细胞为研究对象，用基因芯片技术、RNAi技术、基因过表达技术、逐级信号阻断等手段，探索中药对VEGFR-2介导的Ras/MAPK和PI3K/AKT信号通路cross-talk的影响，阐明补肾健脾中药改善RSA母胎界面血管生成障碍的分子生物学机制，为临床治疗提供思路和方法，丰富“胞脉系于肾”的科学内涵。

母胎界面血管生成障碍是复发性流产的重要因素之一。补肾安胎冲剂以补肾健脾立法，有明确的改善母胎界面血管生成的作用。本课题在前期研究的基础上，以母胎界面血管生成为主线，通过建立RSA小鼠模型，含药血清体外人细胞的培养，探索中药对血管生成的调控及VEGFR2介导的Ras/MAPK和PI3K/AKT信号通路串话的影响，从多途径、多靶点，对补肾安胎冲剂改善母胎界面血管生成障碍的分子生物学机制进行研究。.研究内容及结果如下：1.补肾安胎冲剂可降低胚胎丢失率；2. 通过ELISA法，补肾安胎冲剂可提高孕鼠外周血E2、P、VEGF、HIF-1、TGF-β、Ras、MAPK、PI3K、AKT含量，降低VHL、sFlt－1 含量；3.通过PCR法、WB和蛋白印迹法，补肾安胎冲剂可上调孕鼠蜕膜组织中EP、PR、VEGF、VEGFR2、HIF-1、TGF-β的 mRNA水平及蛋白表达，下调VHL、sFlt－1的mRNA水平及蛋白表达；4.通过免疫组化和蛋白印迹法，补肾安胎冲剂可上调孕鼠蜕膜组织中AKT、PI3K、Ras、MARK蛋白表达；5.通过蛋白印迹法，补肾安胎冲剂上调孕鼠蜕膜组织中MVD蛋白表达；6.通过提取孕鼠胎盘微血管内皮细胞、平滑肌细胞、成纤维细胞，运用免疫荧光和蛋白印迹法，补肾安胎冲剂上调细胞中AKT、PI3K、Ras、MARK、VEGF、VEGFR2蛋白表达；7.通过对含药血清人体外细胞培养，运用沉默技术，证实PI3K/AKT及RAS/MAPK信号通路在体外胎盘微血管内皮细胞中存在着串话；运用PCR、WB等法，补肾安胎冲剂含药血清可改善转染了 VEGFR-2 SiRNA的胎盘微血管内皮细胞VEGF、VEGFR2、Ras、MAPK、PI3K、AKT mRNA及VEGF、VEGFR2、Ras、MAPK、P-MAPK、PI3K、AKT、P-AKT蛋白表达水平。.结论及科学意义：补肾安胎冲剂可降低流产率及胚胎丢失率；其促进母胎界面血管生成，降低流产率的机制是，调控血管生成的正负调节因子，促进胎盘微血管内皮细胞的增殖、迁移和成管，通过VEGF介导的RAS/MAPK、PI3K/AKT信号通路对血管生成进行调节，促进胎盘血管新生；并首次从细胞学角度，证实了PI3K/AKT及RAS/MAPK信号通路在体外胎盘微血管内皮细胞中存在着串话，阐明了补肾健脾中药改善母胎界面血管生成障碍的作用。