基于NK/M-CSF/NO通路的二补助育汤调节子宫内膜容受性效应机制相关性研究

Erbuzhuyu decoction is the unique experienced prescription of the senior TCM Professor Xiao Chengcong of low endometrial receptivity treatment, early clinical studies have shown it can significantly improve the receptivity of the endometrium, and promote the success rate of IVF-ET pregnancy.Early experimental studies have shown that traditional Chinese medicine may be involved in the adhesion process of embryoim plantation by SCF, LIF, ER, PR, ανβ3, HOXA-10,so as to affect endometrial receptivity. To clarify further research on the position adhesion,and invasion on embryo implantation, we suppose NK/M-CSF /NO pathway, using mouse models made by indomethacin solution, on nitrate reductase method, on Real-time PCR method and flow cytometry instrument to research Erbuzhuyu decotion's influence on NK / M-CSF / NO pathway and pinopode expression. To clarify the targets effect and overall mechanisms on behalf of the TCM prescriptions to improve the pregnancy rate of IVF-ET，and figure out the mechanism of "nourishing the Qi of kidney with filling up the vessels of uterus" which may provide the experimental and theoretical basis for clinical improve the pregnancy rate in IVF-ET.

二补助育汤是名老中医肖承悰教授治疗子宫内膜容受性低的独特经验方，前期临床研究表明，其能明显改善子宫内膜容受性，提高再次IVF-ET妊娠成功率；前期实验研究表明，中药可能通过SCF、LIF、ER、PR、整合素ανβ3、HOXA-10等参与胚胎植入的黏附过程，从而影响子宫内膜容受性。为进一步阐明中药对胚胎植入定位、黏附、侵入过程的影响，我们提出NK/M-CSF/NO通路，采用吲哚美辛方案制备动物模型，应用硝酸还原酶法、Real-time PCR、流式细胞仪等技术，研究二补助育汤对子宫内膜该通路及胞饮突表达的影响，从而进一步揭示以"补肾气，通胞脉"立法之中药提高子宫内膜容受性的作用靶点和整体效应机制，为临床提高IVF-ET受孕率提供实验及理论依据。

二补助育汤是名老中医肖承悰教授治疗子宫内膜容受性低的经验方，前期临床研究表明其能改善患者临床症状，提高再次IVF-ET成功率；前期实验研究表明其能通过胞饮突、SCF、ανβ3、HOXA-10等靶点参与胚胎定位、黏附等过程。本研究采用吲哚美辛方案造成胚胎着床障碍动物模型，于pd5取材并计数胚泡着床数目，并于扫描电镜下观察小鼠子宫内膜胞饮突的数目与形态，且分别采用流式细胞技术、Real-time PCR技术、硝酸还原酶法检测不同剂量二补助育汤、补佳乐、阿司匹林作用下小鼠子宫内膜NK细胞含量、M-CSF mRNA表达及含量、NO含量，从而探究二补助育汤对子宫内膜NK/M-CSF/NO通路及形态学的影响。结果表明，二补助育汤能够增加子宫内膜厚度，促进腺体分泌以及血管的生成，提高细胞的能量代谢和蛋白合成，促进胞饮突的表达，且提高了子宫内膜M-CSF mRNA表达量及NO含量。由于各组间NK细胞含量差异不甚明显，而NK细胞与子宫内膜血管生成关系密切，故又采用Real-time PCR、Western blot、免疫组化法分别检测各组小鼠子宫内膜PI3K、Akt、eNOS基因的表达、蛋白的表达及定位，并采用Real-time PCR检测VEGF mRNA的表达，从而探究二补助育汤对与血管生成相关的PI3K/Akt/eNOS信号通路及其上游信号分子VEGF的影响。结果表明，各组eNOS mRNA表达无显著差异；其中二补助育汤低剂量组PI3K mRNA、Akt mRNA表达较模型组有显著差异，低剂量组PI3K mRNA表达明显高于补佳乐组和阿司匹林组，Akt mRNA表达与补佳乐组和阿司匹林组无显著差异；二补助育汤高剂量组p-Akt蛋白表达量显著高于补佳乐组、阿司匹林组，二补助育汤各剂量组p-PI3K、p-eNOS蛋白量与补佳乐组、阿司匹林组无显著差异；二补助育汤对提高子宫内膜VEGF表达的效果较其他各组更加明显。综上，该课题以名老中医验方二补助育汤入手，揭示中医药提高子宫内膜容受性的作用靶点和效应机制，为临床提高IVF-ET受孕率提供实验及理论依据。