【作者】 王波；

【导师】 靳镭； 王世宣；

【作者基本信息】 华中科技大学 ， 妇产科学（专业学位）， 2022， 博士

【摘要】 研究目的顺铂作为一线抗肿瘤药物,可导致女性生殖系统发育停滞、早绝经和生育力丧失等,但其机制尚未完全阐明,且缺乏有效干预策略。本研究拟通过单细胞转录组测序揭示顺铂暴露小鼠卵巢细胞组成和基因表达变化。在此基础上,探讨二甲双胍对顺铂暴露小鼠卵巢储备和功能的保护效应并绘制单细胞转录组图谱,旨在明确二甲双胍通过重编程卵巢各类细胞及其基因表达对抗顺铂所致卵巢损伤的潜在机制。研究方法选取有正常动情周期的雌性C57BL/6小鼠120只(随机分为4组,N=30只/组)。对照组(Con)给予生理盐水灌胃和腹腔注射处理;顺铂组(Cis)给予5.0mg/kg剂量顺铂腹腔注射一次;顺铂组+二甲双胍组(Cis+Met)给予相同剂量顺铂暴露,二甲双胍(250mg/kg)灌胃预处理1周且持续至实验结束;二甲双胍组(Met)给予相同剂量和周期灌胃处理。期间,监测各组小鼠一般情况及动情周期,收集小鼠卵巢及血清标本,进行卵泡计数和激素水平检测以评估小鼠卵巢功能。与此同时,对捕获的小鼠卵巢单细胞进行聚类分析,包括对各类细胞数量和占比、各细胞基因和转录因子表达进行分析;采用单细胞拟时序算法,构建并分析不同卵泡发育阶段的颗粒细胞分化轨迹;整合卵巢各细胞配体-受体信息,基于Cell Phone DB预测卵巢各细胞之间的通讯关系,采用共培养体系培养小鼠卵巢原代颗粒细胞及RAW264.7巨噬细胞系,进行体外实验验证。研究结果1.顺铂暴露的小鼠一般情况差,规律动情周期小鼠比例减少,血清FSH水平上升、E2水平下降,卵巢纤维化程度加重;二甲双胍可以改善顺铂暴露的小鼠动情周期及FSH、E2表达水平,逆转卵巢组织纤维化。2.顺铂暴露的小鼠卵巢各细胞数量发生显著变化,其中T细胞、浆细胞、单核细胞、始基卵泡颗粒细胞及淋巴管内皮细胞占比下降,而其他卵巢体细胞占比上升;二甲双胍可以通过正向逆转免疫细胞(T细胞、浆细胞、单核细胞、树突状细胞)、卵泡颗粒细胞及淋巴管内皮细胞占比,负向逆转其他细胞占比,调节卵巢细胞微环境。3.顺铂可上调包括免疫细胞趋化、细胞因子分泌、凋亡和氧化应激等基因和转录因子表达;二甲双胍可以逆转促炎基因表达,并通过调节分泌细胞因子的免疫细胞与其他细胞的交互(配体-受体互作关系),减少卵巢内炎症因子分泌,改善卵泡生长发育的炎症微环境。同时,二甲双胍对凋亡、氧化应激、卵巢纤维化等通路和基因亦有正向调控作用。4.顺铂暴露引起小鼠卵巢各级卵泡颗粒细胞(始基卵泡颗粒细胞、卵丘颗粒细胞)凋亡;拟时序分析结果显示,顺铂会加速始基卵泡激活,干扰从始基卵泡至生长卵泡的正常分化发育轨迹;二甲双胍可以减少卵丘颗粒细胞凋亡,逆转卵泡的分化发育轨迹,并且可以募集M2型巨噬细胞趋化至闭锁卵泡,对颗粒细胞发挥“胞葬”效应,清除炎症细胞因子诱导的凋亡颗粒细胞。研究结论1.二甲双胍可以改善顺铂暴露的小鼠卵巢储备、内分泌功能及纤维化。2.绘制了二甲双胍对抗顺铂所致卵巢损伤的单细胞转录组图谱,揭示了顺铂损伤卵巢各类细胞组成、基因表达及细胞间的通讯。在此基础上,明确二甲双胍可以重编程顺铂暴露小鼠的卵巢各类细胞基因、转录因子及细胞互作。3.二甲双胍可以调控卵巢各类细胞分泌的细胞因子,消除顺铂暴露小鼠卵巢各细胞之间促炎因子积累,改善卵巢免疫炎症微环境。更多还原

【Abstract】 Objective Cisplatin is a first-line antitumor drug,which can lead to early menopause and loss of fertility in women.However,the mechanism of cisplatin-induced ovarian damage has not been fully clarified,and there is a lack of effective intervention strategies.This study aims to reveal the changes in ovarian cell composition and gene expression after cisplatin intervention by single-cell transcriptome sequencing.On this basis,to explore the protective effect of metformin on cisplatin-induced ovarian reserve and function in mice,draw the single-cell transcriptome atlas,and clarify the potential mechanism of metformin against cisplatin-induced ovarian damage by reprogramming various ovarian cells and their gene expression.Methods120 female C57BL/6 mice with normal estrous cycle were selected(randomly divided into4 groups,N=30/group).The control group(Con)was treated with normal saline by gavage and intraperitoneal injection;the Cisplatin group(Cis)was given 5.0mg/kg cisplatin intraperitoneal injection once;Cisplatin group + metformin group(Cis +Met)was treated with the same dose of cisplatin.Metformin(250mg/kg)was pretreated by gavage for one week and continued until the end of the experiment;Metformin group(Met)was given the same dose and cycle by gavage.During this period,the general condition and estrous cycle of mice in each group were monitored,the ovarian and serum samples of mice were collected,and the follicular count and hormone level were detected to evaluate the ovarian function of mice.At the same time,cluster analysis was carried out on the captured mouse ovarian single cells,including the number and proportion of various cells,the expression of genes and transcription factors in each cell;The granulosa cell differentiation trajectories of different follicular development stages were constructed and analyzed by single-cell quasi temporal algorithm;Integrate the ligand-receptor information of ovarian cells,predict the communication relationship between ovarian cells based on cellphone DB,and use the co-culture system to culture mouse ovarian primary granulosa cells and RAW264.7 macrophage lines were tested in vitro.Results1.The general condition of mice intervened by cisplatin was poor,the proportion of mice in regular estrous cycle decreased,the level of serum FSH increased,the level of E2 decreased,and the degree of ovarian fibrosis increased;Metformin can improve the estrous cycle and the expression levels of FSH and E2 in mice intervened by cisplatin,and reverse ovarian fibrosis.2.The number of ovarian cells after cisplatin intervention changed significantly,in which the proportion of T cells,plasma cells,monocytes,primordial follicular granulosa cells,and lymphatic endothelial cells decreased,while the proportion of other ovarian somatic cells increased;Metformin can positively reverse the proportion of immune cells(T cells,plasma cells,monocytes,dendritic cells),follicular granulosa cells and lymphatic endothelial cells,and negatively reverse the proportion of other cells to regulate the microenvironment of ovarian cells.3.Cisplatin up-regulated the expression of genes and transcription factors including immune cell chemotaxis,cytokine secretion,apoptosis,and oxidative stress;Metformin can reverse the expression of pro-inflammatory genes,reduce the secretion of inflammatory factors in the ovary and improve the inflammatory microenvironment of follicular growth and development by regulating the interaction between immune cells secreting cytokines and other cells(ligand-receptor interaction).At the same time,metformin had a positive regulatory effect on apoptosis,oxidative stress,ovarian fibrosis,and other related pathways and genes.4.Cisplatin induced apoptosis of follicular granulosa cells(primordial follicular granulosa cells and cumulus granulosa cells);Pseudo time series analysis showed that cisplatin could accelerate the activation of primordial follicles and interfere with the normal differentiation and development track from primordial follicles to growing follicles;Metformin can reduce the apoptosis of cumulus granulosa cells,reverse the differentiation and development trajectory of follicles,recruit M2 macrophages to chemotaxis to atresia follicles,exert the "cell burial" effect on granulosa cells and reduce the apoptosis of granulosa cells induced by inflammatory cytokines.Conclusion1.Metformin attenuates cisplatin-induced ovarian reserve,endocrine function and alleviates fibrosis in mice.2.The single cell transcriptome map of metformin against cisplatin induced ovarian injury is drawn,which reveals the changes of cell composition,gene expression and intercellular communication of cisplatin induced ovarian damage.On this basis,it is clear that metformin can reprogram the genes,transcription factors and cell interactions of ovarian cells intervened by cisplatin.3.Metformin can regulate the cytokines secreted by various ovarian cells,eliminate the accumulation of proinflammatory factors among ovarian cells,and improve the immune and inflammatory microenvironment of the ovary.更多还原