自噬相关蛋白Atg5及P62在DEHP引起新生鼠卵泡发育异常过程中的作用

Regulation of Atg5 and P62 during the follicular dysplasia process of newborn mice induced by DEHP

目的：探讨自噬相关蛋白Atg5及P62在邻苯二甲酸二乙基己酯（diethylhexyl phthalate,DEHP）引起新生鼠卵巢卵泡发育异常这一过程中的作用。方法：将健康6-8周龄清洁级成年雌性昆明鼠随机分为对照组（玉米油）和DEHP组,每组30只,均不做任何处理。成年雌鼠生产后的新生鼠分为对照组（玉米油）和DEHP组,采用腹腔注射方式,DEHP组按0.62 mg/kg浓度对新生鼠进行注射,对照组注射相同体积玉米油,从新生鼠出生第1天开始注射,每天1次,连续5 d,注射结束后收集新生鼠卵巢组织,HE染色观察卵巢各级卵泡的发育情况,q RT-PCR及Western blot检测卵泡发育标志分子GDF9及自噬相关蛋白Atg5、P62的表达情况。结果：与对照组比较,HE染色结果显示DEHP组原始卵泡数量明显减少（P=0.005）,而初级卵泡数量无明显改变（P=0.555）;q RT-PCR结果显示DEHP组新生鼠卵巢组织中卵泡发育标志分子GDF9 m RNA表达明显降低（P=0.034）,而自噬标志分子Atg5、P62 m RNA表达明显增加（P值分别为0.001、0.024）。Western blot结果显示DEHP组新生鼠卵巢中自噬相关蛋白Atg5及P62均明显增加,差异有统计学意义（P值分别为0.008、0.001）。结论：在DEHP暴露引起新生鼠卵巢卵泡发育异常这一过程中,自噬标记蛋白Atg5及P62可能发挥着重要作用。

Objective：To investigate the effects of autophagy related protein Atg5 and P62 on the process of newborn mice follicu- lar dysplasia caused by DEHP. Methods： Adult female Kun Ming mice of six to eight weeks at clean and healthy level were randomly divided into the control groups（corn oil） and the DEHP groups（30 in each group） without any treatment. The newborns were divided into the control groups（corn oil） and the DEHP groups. By intraperitoneal injection,the newborns of the DEHP groups were injected with DEHP at the concentration of 0.62 mg/kg,and the ones of the control groups were injected with the same volume of corn oil. The newborn mice were injected once a day from born to 5 days postpartum. Ovarian tissues of the newborn mice from two groups were collected. HE staining was used to observe the development of ovarian follicles at all levels. Real-time PCR and Western blot were employed to detect the expression of follicular developmental marker GDF9 and autophagy related proteins Atg5 and P62. Results ： Compared with that of control groups, the number of primordial follicles of newborn mice from the DEHP groups decreased sig- nificantly, and there was no significant difference in the primary follicles number of the newborn mice between the two groups. The re- suits of Real-time PCR showed the levels of follicular developmental marker GDF9 decreased significantly,butautophagy related gene Atg5 and P62 mRNA in the newborn mice ovarian tissues from the DEHP group increased significantly. Western blot showedthe expression of autophagy related proteins Atg5 and P62 in the newborn mice ovarian tissues were significantly increased following DEHP treatment. Concltusion：Atg5 and P62 might play an important role during the follicular dysplasia process of newborn mice induced by DEHP.