脱氢表雄酮能促进体外人卵巢颗粒细胞——KGN细胞的凋亡  

Dehydroepiandrosterone can promote the apoptosis of KGN in human ovarian granulosa cells

作　　者：田东梅[1] 李亨利[1] 朱明辉[1] 单旭东 庄静 杨丹[1] Tian Dongmei;Li Hengli;Zhu Minghui;Shan Xudong;Zhuang Jing;Yang Dan(The Second Affiliated Hospital of Chengdu University of Traditional Chinese Medical, Chengdu 610000, China)

机构地区：[1]成都中医药大学第二临床医学院/第二附属医院,610000

出　　处：《中华生殖与避孕杂志》2018年第4期305-310,共6页Chinese Journal of Reproduction and Contraception

基　　金：四川省教育厅重点项目(15ZA0097);四川省教育厅科研创新团队项目(17TD0014)~~

摘　　要：目的观察脱氢表雄酮(dehydroepiandrosterone,DHEA)对体外人卵巢细胞——KGN细胞株凋亡的影响。方法用不同剂量的DHEA(0.01 mmol/L、0.1 mmol/L、1 mmol/L、10 mmol/L)处理体外培养的KGN细胞,以不加DHEA为阴性对照组,然后分别用CCK-8法和流式细胞术检测KGN细胞的活性和凋亡情况;并通过实时荧光定量逆转录-多聚酶链反应(real-time fluorescent quantitative reverse transcriptase polymerase chain reaction,q RT-PCR)检测雄激素受体(androgen receptor,AR)、B淋巴细胞瘤-2(B-cell lymphoma-2,BCL-2)和BCL-2相关X蛋白(BCL-2 associated X protein,BAX)基因m RNA的表达水平;通过蛋白免疫印迹(Western blotting)检测AR、BCL-2、BAX、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)及磷酸化ERK(P-ERK)的表达水平。结果与阴性对照组相比,经DHEA处理的KGN细胞的活力显著下降(P=0.000),而且随着DHEA剂量的增加,KGN细胞的凋亡率也随之提高,其中10 mmol/L DHEA干预组的凋亡率与阴性对照组相比,差异具有统计学意义(P=0.019);用不同浓度DHEA处理KGN细胞后,细胞中BCL-2蛋白的表达水平随着DHEA浓度的增加逐渐降低,其中10 mmol/L DHEA干预组与阴性对照组相比差异有统计学意义(P=0.007);与阴性对照组相比,0.1 mmol/L和10 mmol/L DHEA干预组细胞中BCL-2 m RNA的表达水平也显著降低(P=0.007,P=0.012);而细胞中AR和BAX的表达水平无显著变化(P>0.05);此外,虽然DHEA干预组KGN细胞中ERK蛋白的表达水平与阴性对照组相比,差异无统计学意义(P>0.05),但P-ERK蛋白的表达水平随着DHEA浓度的增加逐渐降低,其中10 mmol/L DHEA干预组细胞中P-ERK蛋白的表达水平显著降低(P=0.005)。结论 DHEA能抑制KGN细胞的生长而促进其凋亡,并伴随着细胞中BCL-2蛋白和P-ERK蛋白表达水平的下降。Objective To observe the effect of dehydroepiandrosterone （DHEA） on the apoptosis of KGN cell lines.Methods KGN cell line were treated with DHEA at different concentrations （0.01 mmol/L, 0.1 mmol/L, 1 mmol/L, 10 mmol/L）. The activity and apoptosis of KGN cells were detected by CCK-8 assay and flow cytometry respectively. Real-time fluorescent quantitative reverse transcriptase polymerase chain reaction （QRT-PCR） was used to detect the expression of androgen receptor （AR）, B-cell lymphoma-2 （BCL-2） and BCL-2 associated X protein （BAX） gene mRNA expression levels. The expression of AR, BCL-2, BAX, extracellular regulated protein kinases （ERKs） and phosphorylated ERKs （P-ERKs） were detected by Western blotting.Results Compared with negative control group, cell viability of KGN treated with DHEA decreased significantly （P=0.000）, and the apoptosis rate of KGN cells increased with the increase of DHEA dose. The apoptosis of KGN treated with 10 μmol/L DHEA was significantly different from that in negative control group （P=0.019）. The expression of BCL-2 protein gradually decreased with the increasing concentration of DHEA. Differences between DHEA 10 mmol/L group and the negative control group were statistically significant （P=0.007）. Compared with control group, the expression of BCL-2 mRNA in the cells treated with 0.1 mmol/L and 10 mmol/L DHEA was significantly decreased （P=0.039, P=0.012）, while the expression of AR and BAX in the cells did not change significantly （P〉0.05）. In addition, although the expression level of ERK protein in KGN cells was not significantly different from that in the negative group （P〉0.05）, the expression of P-ERK protein decreased gradually with the increase of DHEA concentration. The expression of P-ERK protein in 10 mmol/L DHEA-treated group was significantly decreased （P=0.005）.Conclusion DHEA can inhibit the growth of KGN cells and promote its apoptosis with the decrease of BCL-2 and P-ERK protein expression, suggesting

关 键 词：脱氢表雄酮(DHEA) KGN细胞 细胞凋亡 B淋巴细胞瘤-2(BCL-2) 磷酸化细胞外调节蛋白激酶(P-ERK) 

分 类 号：R339.22[医药卫生—人体生理学]