慢病毒载体介导的类固醇生成因子-1沉默调控子宫内膜基质细胞蜕膜化与自噬  

Lentiviral vector mediated steroidogenic factor-1 silencing regulating decidualization and autophagy of endometrial stromal cells

作　　者：任健 陈晓燕[3] REN Jian;CHEN Xiao-yan(School of Medicine,Jiangsu University Pathology Teaching and Research Office,Jiangsu Zhenjiang 212000,China;Department of Cardiopulmonary Rehabilitation,Wuxi Rehabilitation Hospital,Jiangsu Wuxi 214000,China;Department of Gynecology,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400000,China)

机构地区：[1]江苏大学医学院病理学教研室,江苏镇江212000 [2]无锡市康复医院心肺康复科,江苏无锡214000 [3]重庆市中医院妇产科,重庆400000

出　　处：《解剖学报》2024年第2期188-195,共8页Acta Anatomica Sinica

基　　金：重庆市自然科学基金(cstc2019jcyj-msxmX0749)。

摘　　要：目的探讨慢病毒载体介导的类固醇生成因子-1(SF-1)沉默对人子宫内膜基质细胞(hESCs)蜕膜化进程的影响及机制。方法收集子宫内膜异位症(EM)患者与正常妊娠者的子宫内膜组织,通过Real-time PCR和免疫组织化学染色检测SF-1的差异表达情况。从正常妊娠者的子宫内膜组织中分离并鉴定hESCs,将hESCs分为对照组、雌二醇(E_(2))+孕酮(P4)组、短发夹RNA(shRNA,sh)-正常对照(NC)+E_(2)+P4组、sh-SF-1+E_(2)+P4组,进行对应处理后,倒置显微镜下观察hESCs形态变化,Real-time PCR和Western blotting分别检测细胞内人胰岛素样生长因子结合蛋白1(IGFBP-1)、泌乳素(PRL)的mRNA表达水平和蛋白表达水平,流式细胞术测定细胞周期分布,免疫荧光染色观察细胞内自噬标记物微管相关蛋白轻链3(LC3)表达情况,Western blotting测定细胞内自噬相关蛋白LC3-Ⅱ、LC3-Ⅰ、Beclin-1表达水平。结果EM患者子宫内膜组织中SF-1 mRNA相对表达量和SF-1蛋白阳性率均高于正常妊娠者子宫内膜组织(P<0.05)。与sh-NC+E_(2)+P4组比较,sh-SF-1+E_(2)+P4组细胞多为长梭形,未见明显蜕膜化,IGFBP1、PRL的mRNA相对表达量与蛋白相对表达量均显著下调(P<0.05),G_(0)/G_(1)期细胞比例显著减少而S期细胞比例显著增加(P<0.05),细胞内LC3荧光表达增强,LC3-Ⅱ/LC3-Ⅰ比值显著升高,Beclin-1蛋白相对表达量也显著上调(P<0.05)。结论EM患者子宫内膜组织内SF-1表达升高,通过慢病毒载体介导的SF-1沉默能够抑制hESCs蜕膜化过程,该作用可能与调控细胞自噬水平相关。Objective To explore the influence and mechanism of lentiviral vector-mediated steroidogenic factor-1(SF-1)silence on decidual process of human endometrial stromal cells(hESCs).Methods The endometrial tissues of patients with endometriosis(EM)and normal pregnant women were collected,and the differential expression of SF-1 was detected by Real-time PCR and immunohistochemical staining.hESCs were isolated from the endometrial tissue of normal pregnancy and identified,hESCs were divided into control group,estradiol(E_(2))+progesterone(P4)group,short hairpin RAN(shRNA,sh)-normal control(NC)+E_(2)+P4 group,sh-SF-1+E_(2)+P4 group,after the corresponding processing,the morphological changes of hESCs were observed under an inverted microscope.Real-time PCR and Western blotting were used to detect the mRNA expression levels and protein levels of human insulin-like growth factor-binding protein-1(IGFBP-1)and prolactin(PRL)in cells,respectively,flow cytometry was used to determine the cycle distribution of the cells,immunofluorescence staining was used to observe the expression of the intracellular auto-bid microtubule-associated protein light chain 3(LC3),Western blotting was used to determine the protein level of intracellular autophagy-related proteins LC3-Ⅱ,LC3-Ⅰ and Beclin-1.Results The relative expression of SF-1 mRNA and the positive rate of SF-1 protein in endometrial tissue of EM patients were higher than those of normal pregnancy endometrial tissue(P<0.05).Compared with the sh-NC+E_(2)+P4 group,the cells in the sh-SF-1+E_(2)+P4 group were mostly long-spindle,and there was no obvious decidualization.The relative mRNA expression and protein expression of IGFBP1 and PRL were significantly downregulated(P<0.05),the proportion of cells in G_(0)/G_(1) phase was significantly decreased,the proportion of cells in S phase was significantly increased(P<0.05),the ratio of LC3-Ⅱ/LC3-Ⅰ was significantly increased,and the relative expression of Beclin-1 protein was also significantly up-regulated(P<0.05).Conclusion The

关 键 词：子宫内膜基质细胞 类固醇生成因子-1 蜕膜化 自噬 免疫印迹法 人 

分 类 号：R711[医药卫生—妇产科学]