摘要
目的研究腹膜型Ⅰ~Ⅱ期子宫内膜异位症(endometriosis,EMS)患者腹腔液与肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)对体外培养的颗粒细胞的功能和自噬的影响,从而探讨腹膜型EMS的不孕原因。方法体外培养人卵巢颗粒细胞系KGN,分别加入EMS患者腹腔液(EMS组)和重组TNF-α蛋白(浓度分别为1μg/L、10μg/L、100μg/L)(TNF-α组)处理细胞,以非EMS患者腹腔液作对照组。采用CCK-8法检测细胞增殖水平,流式细胞术和线粒体膜电位检测细胞凋亡,酶联免疫吸附试验(ELISA)检测培养液雌二醇和孕酮水平,蛋白质印迹法检测自噬标志物LC3-Ⅱ、LC3-Ⅰ和p62,透射电子显微镜观察自噬小体数量。结果加入EMS患者腹腔液可抑制KGN颗粒细胞增殖(P<0.001),促进颗粒细胞凋亡(P=0.009),减少雌、孕激素的分泌(P<0.001,P=0.005);EMS患者腹腔液和高浓度重组TNF-α蛋白可促进颗粒细胞自噬标志物LC3-Ⅱ/LC3-Ⅰ比值上升(P<0.001),p62蛋白减少(P<0.05),自噬体数量增加(P<0.001),自噬过度激活。结论EMS患者腹腔液和TNF-α通过抑制卵巢颗粒细胞增殖、促进凋亡,影响雌、孕激素的分泌和过度自噬等多个环节影响颗粒细胞的功能,进而可能通过颗粒细胞的旁分泌和自分泌功能影响卵子质量、受精及随后的胚胎发育和胚胎着床,从而导致EMS患者不孕。
Objective To investigate the effects of peritoneal fluid of peritoneal type I-II endometriosis(EMS)patients and tumor necrosis factor-α(TNF-α)on granulosa cells function and autophagy,and to investigate the causes of infertility in peritoneal endometriosis.Methods Human ovarian granulosa cell line KGN was cultured in vitro,and cells were treated with peritoneal fluid of EMS patients(EMS group)and recombinant TNF-αprotein in different concentrations(1μg/L,10μg/L,100μg/L)(TNF-αgroup),respectively.Peritoneal fluid of non-EMS patients served as control group.Cell proliferation was detected by CCK-8 method,cell apoptosis was detected by flow cytometry and mitochondrial membrane potential,estradiol and progesterone levels were detected by enzyme-linked immunosorbent assay(ELISA),and autophagic markers including LC3-II/LC3-I and p62 were detected by Western blotting.The number of autophagosomes was observed by transmission electron microscopy.Results EMS patients’peritoneal fluid and high concentrations of recombinant TNF-αprotein inhibited KGN granulosa cell proliferation(P<0.001),promoted granulosa cell apoptosis(P=0.009),and decreased estradiol and progesterone secretion levels(P<0.001,P=0.05),promoted granulocyte autophagy markers LC3-II/LC3-I ratio increased(P<0.001),the p62 protein decreased(P<0.05),the number of autophagosomes increased(P<0.001),and autophagy was over activated.Conclusion EMS patients’peritoneal fluid and TNF-αcan affect the function of granulosa cells by inhibiting the proliferation,promoting apoptosis,affecting the secretion of estradiol and progesterone,and causing excessive autophagy.The paracrine and autocrine functions of granulosa cells may affect oocyte quality,fertilization and embryo development and implantation,resulting in infertility in EMS patients.
作者
钟方圆
谢青贞
周小丹
杨一凡
张祎明
Zhong Fangyuan;Xie Qingzhen;Zhou Xiaodan;Yang Yifan;Zhang Yiming(Center for Reproductive Medicine,Renmin Hospital of Wuhan University,Hubei Clinic Research Center for Assisted Reproductive Technology and Embryonic Development,Wuhan 430060,China)
出处
《中华生殖与避孕杂志》
CAS
CSCD
北大核心
2019年第12期973-980,共8页
Chinese Journal of Reproduction and Contraception
基金
国家自然科学基金面上项目(81471456)。
