摘要
目的 验证多囊卵巢综合征(PCOS)小鼠卵巢颗粒细胞炎症状态,并探索慢性低度炎症状态对PCOS排卵障碍的作用机制。方法 选取SPF级健康雌性C57BL/6小鼠10只,21~25 d,14~16 g,按照随机数字表法将其分为CONTROL组和PCOS组,每组5只。PCOS组每日皮下注射脱氢表雄酮(6 mg/100 g,溶剂:玉米油与DMSO混合液),CONTROL组小鼠每日皮下注射等体积溶剂,连续给药21 d。苏木精-伊红染色观察两组小鼠卵巢组织形态,免疫组织化学染色法检测两组小鼠卵巢颗粒细胞中肿瘤坏死因子-α(TNF-α)表达水平。向KGN细胞中加入脂多糖(LPS)0.1μg/ml(LPS 0.1组)、0.2μg/ml(LPS 0.2组)、0.5μg/ml(LPS 0.5组)、1.0μg/ml(LPS 1.0组)、5.0μg/ml(LPS 5.0组)构建颗粒细胞炎症模型,以KGN细胞作为对照组。Western blot法检测KGN细胞中TNF-α、SMAD4表达水平;免疫荧光检测对照组及LPS 0.2组KGN细胞中YTHDF2表达水平。转染PC3.1-YTHDF2质粒构建YTHDF2过表达颗粒细胞模型(PC3.1-YTHDF2组),转染PC3.1质粒作为对照(PC3.1组)。Western blot法检测PC3.1组和PC3.1-YTHDF2组KGN细胞中SMAD4表达水平。结果 CONTROL组小鼠卵巢中各时期卵泡均存在。与CONTROL组比较,PCOS组小鼠卵巢中存在大量未成熟卵泡。与CONTROL组比较,PCOS组小鼠卵巢颗粒细胞中TNF-α表达水平升高(P<0.05)。与对照组比较,LPS 0.2组、LPS 0.5组、LPS 1.0组、LPS 5.0组KGN细胞中TNF-α表达水平升高(P<0.05);LPS 0.1组、LPS 0.2组、LPS 0.5组、LPS 1.0组、LPS 5.0组KGN细胞中SMAD4表达水平降低(P<0.05)。与对照组比较,LPS 0.2组KGN细胞中YTHDF2表达水平升高(P<0.05)。与PC3.1组比较,PC3.1-YTHDF2组KGN细胞中SMAD4表达水平降低(P<0.05)。结论 PCOS小鼠颗粒细胞中存在炎症状态,PCOS颗粒细胞的炎症状态可影响其排卵功能,PCOS颗粒细胞炎症状态可能通过调控YTHDF2影响SMAD4的表达从而造成PCOS排卵障碍。
Objective To verify the inflammatory status of ovarian granulosa cells in polycystic ovary syndrome(PCOS)mice,and to investigate the mechanism of chronic low-grade inflammatory status on ovulation disorders of PCOS.Methods Ten SPF healthy female C57BL/6 mice,21-25 d,14-16 g were selected,and they were divided into CONTROL group and PCOS group according to random number table method,with five mice in each group.The PCOS group received daily subcutaneous injection of dehydroepiandrosterone(6 mg/100 g,solvent:a mixture of corn oil and DMSO),while CONTROL group received daily subcutaneous injection of an equal volume of solvent,the drug was administered continuously for 21 d.Hemotoxylin-eosin staining was used to observe the morphology of ovarian tissue in two groups of mice,and the expression level of tumor necrosis factor-α(TNF-α)in ovarian granulosa cells of mice in both groups was detected by immunohistochemical staining.Lipopolysaccharide(LPS)0.1μg/ml(LPS 0.1 group),0.2μg/ml(LPS 0.2 group),0.5μg/ml(LPS 0.5 group),1.0μg/ml(LPS 1.0 group),and 5.0μg/ml(LPS 5.0 group)were added to KGN cells,granulosa cell inflammation model was constructed,and KGN cells were used as control group.The expression levels of TNF-αand SMAD4 in KGN cells were detected by Western blot;the expression levels of YTHDF2 in KGN cells in control group and LPS 0.2 group were detected by immunofluorescence.YTHDF2-overexpressed granulosa cell model was constructed by transfecting PC3.1-YTHDF2(PC3.1-YTHDF2 group),and transfected PC3.1 plasmid as control(PC3.1 group).The expression levels of SMAD4 in KGN cells in PC3.1 group and PC3.1-YthDF2 group were detected by Western blot.Results Follicles in the ovaries were present of CONTROL group mice at all stages.Compared with CONTROL group,there were a large number of immature follicles in ovaries of PCOS mice.Compared with CONTROL group,the expression level of TNF-αin granulosa cells of mice in PCOS group increased(P<0.05).Compared with control group,the expression levels of TNF-αof KGN cells in LPS 0.2 group,LPS 0.5 group,LPS 1.0 group,and LPS 5.0 group were increased(P<0.05);the expression levels of SMAD4 of KGN cells in LPS 0.1 group,LPS 0.2 group,LPS 0.5 group,LPS 1.0 group,and LPS 5.0 group were reduced(P<0.05).Compared with control group,the expression level of YTHDF2 of KGN cells in LPS 0.2 group was increased(P<0.05).Compared with PC3.1 group,the expression level of SMAD4 of KGN cells in PC3.1-YTHDF2 group was reduced(P<0.05).Conclusion There is an inflammatory state in granulosa cells of PCOS mice,the inflammatory state of granulosa cells in PCOS can affect ovulation function,and the inflammatory state of granulosa cells in PCOS may affect the expression of SMAD4 by regulating YTHDF2,leading to ovulation disorders in PCOS.
作者
夏晴
王大勇
葛方亮
郑菲菲
赵雪
强若男
王文晶
刘雁峰
XIA Qing;WANG Dayong;GE Fangliang;ZHENG Feifei;ZHAO Xue;QIANG Ruonan;WANG Wenjing;LIU Yanfeng(Department of Gynecology,Dongzhimen Hospital,Beijing University of Chinese Medicine,Bejing 100700,China;Basic Medical College,Harbin Medical University,Heilongjiang Province,Harbin 150086,China)
出处
《中国医药导报》
CAS
2024年第14期1-5,共5页
China Medical Herald
基金
国家自然科学基金资助项目(82305298、82274567、82074480)
中国博士后科学基金资助项目(2022M720521)。
