DEHP对小鼠精原细胞Bax和Bcl-2表达水平的影响

邻苯二甲酸(2-乙基己基)酯(di(2-ethylhexyl)phthalate,DEHP)因在环境中的广泛分布而受到普遍关注。为探究DEHP对雄性生殖细胞的毒作用机制,将小鼠精原细胞(GC-1spd)分别暴露于终浓度为0(对照)、10、100、1 000 mg·L~(-1)DEHP的培养基中培养24 h,采用噻唑兰(MTT)法检测细胞相对活力,流式细胞仪检测细胞凋亡情况,实时荧光定量PCR法检测Bax和Bcl-2m RNA的表达情况及Western blot技术检测Bax和Bcl-2蛋白的表达水平。结果显示:在检测细胞相对活力时,1 000 mg·L~(-1)剂量组明显低于其他各剂量组,100 mg·L~(-1)剂量组低于对照组和10 mg·L~(-1)剂量组(P<0.05);在检测细胞凋亡时,1 000 mg·L~(-1)剂量组早期凋亡率高于对照组(P<0.05);晚期凋亡率1 000 mg·L~(-1)和100 mg·L~(-1)剂量组均明显高于对照组,1 000 mg·L~(-1)剂量组高于10 mg·L~(-1)剂量组(P<0.05);总凋亡率呈现明显上升趋势,其中1 000 mg·L~(-1)和100 mg·L~(-1)剂量组明显高于对照组,1 000mg·L~(-1)剂量组还明显高于10 mg·L~(-1)剂量组(P<0.05)。实时荧光定量PCR检测结果显示:Bax m RNA表达水平1 000 mg·L~(-1)和100 mg·L~(-1)剂量组明显高于对照组,1 000 mg·L~(-1)剂量组与10 mg·L~(-1)剂量组比较差异具有统计学意义(P<0.05);Bcl-2 m RNA表达水平1 000 mg·L~(-1)剂量组低于10 mg·L~(-1)剂量组和对照组(P<0.05)。此外,Bax和Bcl-2 m RNA比值1 000 mg·L~(-1)和100 mg·L~(-1)剂量组均明显高于对照组,1 000 mg·L~(-1)剂量组高于10 mg·L~(-1)剂量组(P<0.05)。Western blot结果显示,Bax蛋白表达水平1 000 mg·L~(-1)剂量组高于对照组(P<0.05);Bcl-2蛋白表达水平各剂量组与对照组相比差异均具有统计学意义,且1 000mg·L~(-1)和100 mg·L~(-1)剂量组明显低于10 mg·L~(-1)剂量组和对照组(P<0.05);此外,Bax/Bcl-2两蛋白比值1 000 mg·L~(-1)和100 mg·L~(-1)剂量组明显高于对照组,而1 000 mg·L~(-1)剂量组高于10 mg·L~(-1)剂量组(P<0.05)。结果表明:DEHP可诱导小鼠精原细胞相对活力下降,通过抑制Bcl-2 m RNA和Bcl-2蛋白表达及促进Bax m RNA和Bax蛋白表达实现诱导精原细胞凋亡。

Effect of DEHP on Bax and Bcl-2 Expressions in Mouse Spermatogonial Cells

Di(2-ethylhexyl)phthalate(DEHP) has received wide attention due to its wide distribution in the environment. To explore the toxic mechanism of DEHP on male germ cells, mouse spermatogonial cells(GC-1spd) were exposed to DEHP at the final concentrations of 0 (control), 10, 100, 1 000 mg·L-1DEHP for 24 h. The cell viability was determined by MTT assay, the apoptosis of the mouse spermatogonia measured by flow cytometry, the mRNA expressions of Bax and Bcl-2 genes were detected by real time PCR, and their protein expressions determined by Western blot. The results showed that when detecting the relative activity of cells, the dose of 1000 mg·L-1 was significantly lower than the other groups, the dose of 100 mg·L-1 was lower than the control group and the dose of 10 mg·L-1(P<0.05). When detecting the early apoptotic rates of cells, the dose of 1000 mg·L-1 was higher than the control group(P <0.05); the late apoptosis rate of 1000 mg·L-1 and 100 mg·L-1 dose group were significantly higher than the control group, and 1000 mg·L-1 dose group was higher than 10 mg·L-1(P <0.05); the total apoptosis rate increased obviously, 1000 mg·L-1 and 100 mg·L-1 dose group were significantly higher than the control group, and 1000 mg·L-1 dose group was also higher than 10 mg·L-1(P <0.05). In the quantitative real-time PCR, the expression of Bax mRNA in the doses group of 1000 mg·L-1 and 100 mg·L-1 were significantly higher than the control group, 1000 mg·L-1 and 10 mg·L-1 dose group were statistically significant (P <0.05); the expression of Bcl-2 mRNA in the dose group of 1000 mg·L-1 was lower than the 10 mg·L-1 dose group and the control group (P <0.05); In addition, the ratio of Bax and Bcl-2 mRNA in the doses group of 1000 mg·L-1 and 100 mg·L-1 were significantly higher than the control group, and 1000 mg·L-1 dose group was also higher than 10 mg·L-1. Western blot showed that 1000 mg·L-1 dose group was higher than the control group in the expression of Bax protein (P <0.05). The other groups and the control group were statistically significant, and 1000 mg·L-1 and 100 mg·L-1 dose group were significantly lower than the 100 mg·L-1 dose group and the control group in the expression of Bcl-2 protein (P <0.05). Furthermore, in the ratio of Bax/Bcl-2 protein, 1000 mg·L-1 and 100 mg·L-1 dose group were significantly higher than the control group, and 1000 mg·L-1 dose group was also higher than 10 mg·L-1 (P <0.05). The above results indicate that DEHP could induce the activity of mouse spermatogonia decreased, and inhibite the expression of Bcl-2 mRNA and Bcl-2 protein, and promote the expression of Bax mRNA and Bax protein, which induced the apoptosis of spermatogonial cells.