经血子宫内膜间充质干细胞移植协同补肾调冲方治疗化疗损伤性卵巢早衰的实验研究

Premenopausal women treated with Therarubicin for blood diseases such as lymphoma are at risk for premature ovarian failure (POF).There is no effective treatment for those patients who is eager to have a baby . A new intervention based on Stem cells will be a high potential strategy.Previous research indicate that we have build a rat model for Therarubicin induced POF, and BSTCR may partially improve the microenvironment of ovary.We are supposed to indicate the new mechanism with the intervention of MMCs and BSTCR to the experimental rat models in vitro and in vivo. We will collect the ovarian granulose cells after co-incubated with MMCs and/or BSTCR of at different time points to detect cellular apoptosis and cell cycle with flow cytometry analysis and Western blotting .After applicant MMCs and BSTCR to the rat models in vivo experiment , the rats will be killed at different time points .We perform serum analysis for sexual hormones,ovarian morphology , and test related proteins markers to explore the influence of MMCs and BSTCR to the POF model.

临床发现：淋巴瘤等血液疾病的年轻患者应用吡柔比星治疗后出现卵巢早衰。对迫切恢复女性生育功能的患者，尚无有效治疗。以干细胞为主体的综合干预是新的研究方向。课题前期研究：已构建吡柔比星化疗后卵巢早衰大鼠模型，应用补肾调冲方干预后卵巢微环境有部分改善。本课题拟在前期研究的基础上，应用经血子宫内膜干细胞（MMCs）移植联合补肾调冲方（BSTCR）从体外实验和体内试验两部分探讨干预机制。体外实验：卵巢颗粒细胞与MMCs和/或 BSTCR共同孵育后不同时间点收获，通过流式细胞学分析、Western blot等方法检测凋亡和细胞周期相关蛋白的表达；体内实验：将MMCs单细胞悬液注射至吡柔比星损伤性大鼠模型的卵巢，同时BSTCR灌胃，在不同时间点处死大鼠，取出大鼠血清、卵巢、子宫检测性激素、形态学和相关蛋白的表达，探讨MMCs和BSTCR干预后卵巢形态以及卵巢微环境的变化，为提出MMCs为基础的治疗。

临床发现：淋巴瘤等血液疾病的年轻患者应用表柔吡星治疗后出现卵巢早衰。对迫切要求恢复女性生育功能的患者，尚无有效治疗。以干细胞为主体的综合干预是新的研究方向。本课题应用经血子宫内膜间充质干细胞（MB-MMCs）移植联合补肾调冲方（BSTCR）从体外实验和体内试验两部分探讨干预机制。体外实验：应用贴壁法对志愿者的月经血进行分离、提取及鉴定，证实提取的细胞为MB-MMCs。表柔吡星处理后的卵巢颗粒细胞与MB-MMCs共培养后收获颗粒细胞，通过流式细胞学检测凋亡、ELISA检测性激素分泌水平，MB-MMCs能抑制表柔吡星损伤后的人卵巢颗粒细胞的凋亡，改善其性激素的分泌；体内实验：成功构建表柔吡星损伤性卵巢早衰小鼠模型，经尾静脉将ADVLuc标记的MB-MMCs单细胞悬液注射至表柔吡星损伤性小鼠模型，同时BSTCR灌胃，观察小鼠动情周期恢复后处死小鼠，取小鼠血清检测性激素、观察卵巢形态学、TURNEL检测卵巢颗粒细胞凋亡和Westein blot检测小鼠卵巢bcl-2蛋白的表达，发现BSTCR通过促进MB-MMCs的归巢，协同MB-MMCs，促进小鼠卵巢bcl-2蛋白的表达，抑制小鼠卵巢颗粒细胞的凋亡，促进损伤后小鼠卵巢功能的恢复，本研究提示MB-MMCs联合BSTCR可明显的改善表柔吡星所致卵巢早衰小鼠的卵巢功能。