lncRNA GAS5对胰岛素抵抗模型中卵巢颗粒细胞功能的影响及相关机制研究  

Effects and mechanisms of lncRNA GAS5 on ovarian granulosa cells function in insulin resistance model

作　　者：刘琴[1] 袁蕾 Liu Qin;Yuan Lei(Department of Obstetrics,Shiyan maternal and Child Health Care Hospital,Hubei 442000,China)

机构地区：[1]湖北十堰市妇幼保健院妇产科,十堰442000

出　　处：《中华细胞与干细胞杂志（电子版）》2021年第3期137-145,共9页Chinese Journal of Cell and Stem Cell（Electronic Edition）

摘　　要：目的探究lncRNA GAS5在多囊卵巢综合征(PCOS)中的表达及其对胰岛素抵抗(IR)模型中卵巢颗粒细胞功能的影响及其相关机制。方法收集PCOS包含伴IR者(n=20)与不伴IR者(n=20)及非PCOS患者(n=20)的卵巢颗粒细胞。RT-PCR检测患者颗粒细胞中lncRNA GAS5的表达;应用脂质体稳定转染技术将过表达lncRNA GAS5质粒转染入大鼠卵巢颗粒细胞中,采用地塞米松及胰岛素联合诱导方法建立大鼠颗粒细胞的IR模型;己糖激酶及比色法检测模型中葡萄糖及乳酸的消耗量进行鉴定;将大鼠颗粒细胞分为4组,对照组(正常培养无任何特殊处理的颗粒细胞);IR组(胰岛素抵抗条件下的颗粒细胞);IR-pcDNA3.1-NC组(对转染pcDNA3.1-NC后的颗粒细胞进行IR造模处理);IR-pcDNA3.1-GAS5组(对转染pcDNA3.1-GAS5后的颗粒细胞进行IR造模处理)。应用MTT、Annexin V-FITC、流式细胞术及ELISA实验分别检测过表达lncRNA GAS5对IR条件下颗粒细胞的增殖、凋亡、细胞周期和炎症因子TNF-α、IL-6、IL-18表达的影响;Western blot检测过表达lncRNA GAS5对IR条件下颗粒细胞中磷脂酰肌醇-3(PI3K)/蛋白激酶B(AKT)信号通路p-PI3K、p-AKT及细胞周期蛋白Cyclin B1、CDK1和凋亡相关蛋白Bcl-2、cleaved caspase-3表达的影响。采用重复测量方差分析、独立样本t检验和单因素方差分析比较组间的指标水平差异。结果与对照组比较,IR组和IR-pcDNA3.1-NC组颗粒细胞中lncRNA GAS5的表达(1.09±0.17比0.41±0.11、0.38±0.06)降低,而IR-pcDNA3.1-GAS5组lncRNA GAS5表达水平(1.09±0.17比3.10±0.33)升高,差异均有统计学意义(P均<0.05);与IR组比较,IR-pcDNA3.1-GAS5组颗粒细胞增殖能力(52.16±8.83比68.39±11.14)及细胞周期阻滞(G1期:69.05±10.09比43.51±6.09,S期:25.37±5.59比9.44±2.29,G2/M期:7.11±2.81比47.02±6.69)均升高,细胞凋亡率(33.26﹪±3.44﹪比16.01﹪±2.56﹪),促炎因子TNF-α(138.56±27.05比105.30±12.64)、IL-6(123.69±10.08比86.11±8.03)、IL-18的表达水�Objective To investigate the expression of lncRNA GAS5 in patients with polycystic ovary syndrome(PCOS)as well as the effects and mechanisms of lncRNA GAS5 on the function of ovarian granulosa cells in insulin resistance(IR)model.Methods A total of 60 women were enrolled,and the ovarian granulosa cells were collected,20 of whom with PCOS and IR,20 with PCOS without IR and 20 without PCOS or IR.The expression levels of lncRNA GAS5 in ovarian granulosa cells were detected by RT-PCR.The overexpression of lncRNA GAS5 plasmid was transfected into rat ovarian granulosa cells by liposome stable transfection technology.The IR model of granulosa cells was established by dexamethasone and insulin induction;The consumption of glucose and lactic acid in the model was detected by hexokinase and colorimetry.Rats'granulosa cells were treated with or without insulin,pcDNA3.1-NC and pcDNA3.1-GAS5,and named Control group,IR group,IR-pcDAN3.1-NC group,and IR-pcDAN3.1-GAS5 group,respectively.MTT,Annexin V-FITC,flow cytometry and ELISA were used to detect the effects of lncRNA GAS5 on cell proliferation,apoptosis,cell cycle and the expression of inflammatory factors TNF-α,IL-6 and IL-18 in granulosa cells under IR.Western blot was used to detect the effects of overexpression of lncRNA GAS5 on p-PI3K,p-AKT and cyclin B1 CDK1,Bcl-2 and cleaved caspase-3 in granulosa cells under IR.Two-way repeated-measures ANOVA,t-test and one-way ANOVA were used to compare the differences between the two groups.Results compared with the control group,the expression of lncRNA GAS5 in ovarian granulosa cells of PCOS patients was significantly decreased(1.09±0.17 vs 0.41±0.11,P<0.05),while the expression level of lncRNA GAS5 in granulosa cells of IR model was significantly increased in IR-pcDNA3.1-GAS5 group(1.09±0.17 vs 0.38±0.06,P<0.05).Compared with IR group,granulocytic cell proliferation ability and cell cycle arrest in IR-PCDNA3.1-GAS5 group were(52.16±8.83 vs 68.39±11.14)(G1 phase:69.05±10.09 vs 43.51±6.09),(S phase:25.37±5.59 vs 9.44±2

关 键 词：多囊卵巢综合征 lncRNA GAS5 胰岛素抵抗 卵巢颗粒细胞 

分 类 号：R711.75[医药卫生—妇产科学]