Di(2-ethylhexyl)phthalate(DEHP) has received wide attention due to its wide distribution in the envi-ronment.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 was measured by flow cytome-try,the mRNA expressions of Bax and Bcl-2 genes were detected by real time PCR,and their protein expressions were determined by Western blot. The results showed that when detecting the relative activity of cells, the dose group of 1 000 mg·L-1was significantly lower than the other groups,the group of 100 mg·L-1was lower than the control group and the group of 10 mg·L-1(P<0.05).When detecting the early apoptotic rates of cells,the group of 1 000 mg·L-1was higher than the control group(P<0.05);the late apoptosis rate of 1 000 mg·L-1and 100 mg·L-1 dose group were significantly higher than the control group,and 1 000 mg·L-1dose group was higher than 10 mg ·L-1(P<0.05);the total apoptosis rate increased obviously,1 000 mg·L-1and 100 mg·L-1dose group were signifi-cantly higher than the control group,and 1 000 mg·L-1dose 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 dose groups of 1 000 mg·L-1and 100 mg·L-1 were significantly higher than the control group, and the differences between 1 000 mg·L-1and 10 mg·L-1dose group were statistically significant(P<0.05); the expression of Bcl-2 mRNA in the dose group of 1 000 mg·L-1 was lower than the 10 mg·L-1dose group and the control group(P<0.05);in addition,the ratio of Bax to Bcl-2 mR-NA in the dose groups of 1 000 mg·L-1and 100 mg·L-1were significantly higher than the control group,and 1 000 mg·L-1dose group was also higher than 10 mg·L-1.Western blot showed that 1 000 mg·L-1dose group was higher than the control group in the expression of Bax protein(P<0.05).The differences between other groups and the con-trol group were statistically significant,and 1 000 mg·L-1and 100 mg·L-1dose group were significantly lower than the 10 mg·L-1dose group and the control group in the expression of Bcl-2 protein(P<0.05).Furthermore,in the ratio of Bax/Bcl-2 protein,1 000 mg·L-1and 100 mg·L-1dose group were significantly higher than the control group,and 1 000 mg·L-1dose group was also higher than 10 mg·L-1(P<0.05).The above results indicate that DEHP could de-crease the activity of mouse spermatogonia,and inhibit the expression of Bcl-2 mRNA and Bcl-2 protein,and pro-mote the expression of Bax mRNA and Bax protein,which induced the apoptosis of spermatogonial cells.%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、1000 mg·L-1DEHP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-2 mRNA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,1000 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,1000 mg·L-1剂量组早期凋亡率高于对照组(P<0.05);晚期凋亡率1000 mg·L-1和100 mg·L-1剂量组均明显高于对照组,1000 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1000 mg·L-1和100 mg·L-1剂量组明显高于对照组,1000 mg·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 mRNA表达水平1000 mg·L-1和100 mg·L-1剂量组明显高于对照组,1000 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 mR-NA表达水平1000 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 mRNA比值1000 mg·L-1和100 mg·L-1剂量组均明显高于对照组,1000 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1000 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1000 mg·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1000 mg·L-1和100 mg· L-1剂量组明显高于对照组,而1000 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 mRNA和Bcl-2蛋白表达及促进Bax mRNA和Bax蛋白表达实现诱导精原细胞凋亡.
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