吉林大学学报(医学版) ›› 2024, Vol. 50 ›› Issue (6): 1526-1534.doi: 10.13481/j.1671-587X.20240606

• 基础研究 • 上一篇    

过表达SLC7A5基因对大鼠卵巢颗粒细胞凋亡的影响及其机制

张京顺,邹颖刚,郑连文()   

  1. 吉林大学第二医院生殖中心,吉林 长春 130041
  • 收稿日期:2024-01-08 出版日期:2024-11-28 发布日期:2024-12-10
  • 通讯作者: 郑连文 E-mail:davezheng@sohu.com
  • 作者简介:张京顺(1991-),女,辽宁省抚顺市人,主治医师,医学博士,主要从事多囊卵巢综合征发病机制方面的研究。
  • 基金资助:
    吉林省科技厅自然科学基金项目(20210101268JC)

Effect of over-expression SLC7A5 on apoptosis of ovarian granulosa cells in rats and its mechanism

Jingshun ZHANG,Yinggang ZOU,Lianwen ZHENG()   

  1. Reproductive Center,Second Hospital,Jilin University,Changchun 130041,China
  • Received:2024-01-08 Online:2024-11-28 Published:2024-12-10
  • Contact: Lianwen ZHENG E-mail:davezheng@sohu.com

摘要:

目的 探讨过表达溶质载体家族7成员5(SLC7A5)基因对大鼠卵巢颗粒细胞凋亡的影响,并阐明其相关机制。 方法 4只3周龄SPF级SD雌性大鼠,提取原代大鼠卵巢颗粒细胞,分为阴性对照组(NC组)和促卵泡激素受体(FSHR)染色组(FSHR组)。免疫荧光染色法观察大鼠卵巢颗粒细胞中FSHR蛋白表达情况,鉴定原代大鼠卵巢颗粒细胞是否成功分离。将大鼠卵巢颗粒细胞分为对照组(转染空载体质粒)和OE-SLC7A5组(转染SLC7A5过表达质粒),采用实时荧光定量PCR(RT-qPCR)法和Western blotting法验证细胞转染效率。流式细胞术检测2组卵巢颗粒细胞凋亡率,流式细胞术检测2组不同细胞周期卵巢颗粒细胞百分率,RT-qPCR法检测2组卵巢颗粒细胞中SLC7A5、含半胱氨酸的天冬氨酸蛋白酶(Caspase)-3、Caspase-8和肿瘤坏死因子α(TNF-α) mRNA表达水平,Western blotting法检测2组卵巢颗粒细胞中SLC7A5、Caspase-3、cleaved Caspase-3、Caspase-8、cleaved Caspase-8和TNF-α蛋白表达水平。 结果 荧光显微镜下卵巢颗粒细胞呈长梭形或者不规则形,特异性表达FSHR,NC组未见FSHR绿色荧光表达,FSHR组可见FSHR绿色荧光表达,提示大鼠原代卵巢颗粒细胞成功分离。与对照组比较,OE-SLC7A5组卵巢颗粒细胞中SLC7A5 mRNA和蛋白表达水平均明显升高(P<0.05),提示SLC7A5过表达质粒成功转染卵巢颗粒细胞。流式细胞术检测,与对照组比较,OE-SLC7A5组细胞凋亡率明显升高(P<0.05)。与对照组比较,OE-SLC7A5组S期卵巢颗粒细胞百分率明显降低(P<0.05)。RT-qPCR法检测,与对照组比较,OE-SLC7A5组卵巢颗粒细胞中TNF-α、Caspase-3和Caspase-8mRNA表达水平均明显升高(P<0.05)。Western blotting法检测,与对照组比较,OE-SLC7A5组卵巢颗粒细胞中TNF-α、Caspase-8、cleaved Caspase-8、Caspase-3、cleaved Caspase-3和SLC7A5蛋白表达水平均明显升高(P<0.05)。 结论 SLC7A5蛋白表达增加能够通过上调TNF-α、Caspase-8和Caspase-3凋亡通路表达,促进颗粒细胞凋亡。

关键词: 溶质载体家族7成员5, 大鼠卵巢颗粒细胞, 细胞凋亡, 肿瘤坏死因子α, 含半胱氨酸的天冬氨酸蛋白酶3, 含半胱氨酸的天冬氨酸蛋白酶8

Abstract:

Objective To discuss the effect of over-expression of solute carrier family 7 member 5 (SLC7A5) gene on the apoptosis of ovarian granulosa cells of the rats, and to clarify its related mechanism. Methods Four 3-week-old SPF grade SD female rats were used to extract the primary ovarian granulosa cells of the rats. These cells were divided into negative control group (NC group) and follicle-stimulating hormone receptor (FSHR) staining group (FSHR group). Immunofluorescence staining was used to detect the expressions of FSHR protein in the ovarian granulosa cells of the rats to identify the successful isolation of the primary ovarian granulosa cells of the rats. The ovarian granulosa cells were divided into control group (transfected with empty vector plasmid) and OE-SLC7A5 group (transfected with SLC7A5 over-expression plasmid). Real-time fluoresscence quantitative PCR(RT-qPCR) and Western blotting methods were used to verify the transfection efficiency of the cells; flow cytometry was used to detect the apoptotic rates and cell cycle percentages of the ovarian granulosa cells in two groups; RT-qPCR method was used to detect the expression levels of SLC7A5, cysteinyl aspartate specific proteinase (Caspase)-3, Caspase-8, and tumor necrosis factor-α (TNF-α) mRNA in the ovarian granulosa cells in two groups; Western blotting method was used to detect the expression levels of SLC7A5, Caspase-3, cleaved Caspase-3, Caspase-8, cleaved Caspase-8, and TNF-α proteins in the ovarian granulosa cells in two groups. Results The fluorescence microscope observation results showed that the ovarian granulosa cells appeared spindle-shaped or irregular and specifically expressed FSHR. No FSHR green fluorescence was observed in NC group, while FSHR green fluorescence expression was observed in FSHR group, indicating successful isolation of primary ovarian granulosa cells of the rats. Compared with control group, the expression levels of SLC7A5 mRNA and protein in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased (P<0.05), indicating successful transfection of SLC7A5 over-expression plasmid into the ovarian granulosa cells. The flow cytometry results showed that compared with control group, the apoptotic rate of the cells in OE-SLC7A5 group was significantly increased (P<0.05). Compared with control group, the percentage of the ovarian granulosa cells at S phase in OE-SLC7A5 group was significantly decreased (P<0.05). The RT-qPCR results showed that compared with control group, the expression levels of TNF-α, Caspase-3, and Caspase-8 mRNA in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased (P<0.05). The Western blotting results showed that compared with control group, the expression levels of TNF-α, Caspase-8, cleaved Caspase-8, Caspase-3, cleaved Caspase-3, and SLC7A5 proteins in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased (P<0.05). Conclusion The increased expression of SLC7A5 protein promotes the apoptosis of the granulosa cells by upregulating the expressions of TNF-α, Caspase-8, and Caspase-3 apoptotic pathways.

Key words: Solute carrier family 7 member 5, Ovarian granulosa cell, Apoptosis, Tumor necrosis factor-α, Cysteinyl aspartate specific proteinase-3, Cysteinyl aspartate specific proteinase-8

中图分类号: 

  • R711.75