干细胞因子在新生大鼠卵巢镉暴露致卵母细胞凋亡中的作用  

Effect of stem cell factor on oocyte apoptosis induced by cadmium exposure in neonatal rats

作　　者：罗凌凤[1] 孙义 谢美美[2] 吴停停 刘瑾[1] 张文昌[1] LUO Ling-feng;SUN Yi;XIE Mei-mei;WU Ting-ting;LIU Jin;ZHANG Wen-chang(School of Public Health,Fujian Medical University,Fuzhou Fujian 350122,China;Fujian Provincial Center for Disease Control and Prevention,Fuzhou Fujian 350012,China;Xiamen Children's Hospital,Xiamen Fujian 361006,China)

机构地区：[1]福建医科大学公共卫生学院,福建福州350122 [2]福建省疾病预防控制中心,福建福州350012 [3]厦门市儿童医院,福建厦门361006

出　　处：《毒理学杂志》2023年第3期209-215,共7页Journal of Toxicology

基　　金：国家自然科学基金(81673212)。

摘　　要：目的探讨干细胞因子(stem cell factor,SCF)在新生大鼠卵巢镉暴露致卵母细胞凋亡中的作用。方法在无菌条件下从背部取出4 d龄雌性SD大鼠的卵巢,进行体外培养及染毒处理,染镉(CdCl2)剂量分别为0、0.5、5、10和50μmol/L(μmol/L),连续染毒4 d。为进一步确定镉暴露后卵母细胞凋亡与SCF/c-kit表达的相关性,进一步开展外源性SCF的干预实验,设置空白对照组、100 ng/ml SCF、10μmol/L Cd、10μmol/L Cd+100 ng/ml SCF、10μmol/L Cd+100 ng/ml SCF+2μg/ml ACK-2(c-kit的中和性抗体)组。在透射电子显微镜下观察卵母细胞凋亡超微结构;采用TUNEL法检测卵母细胞凋亡率;采用实时荧光定量PCR法检测卵巢组织中Scf、C-kit、Bax、Bcl-2和Bcl-xl基因的mRNA水平;采用Western blot法检测卵巢组织中Bax、Bcl-2和Bcl-xl蛋白表达水平。结果透射电子显微镜下染毒组可见卵母细胞核皱缩,异染色质增多,可见凋亡小体,线粒体出现空泡、肿胀。与对照组比较,染毒组卵母细胞凋亡率升高,差异有统计学意义(P<0.05);5~50μmol/L组Scf和C-kit mRNA表达水平明显降低,差异有统计学意义(P<0.05);各染毒组Bax mRNA和蛋白表达水平上调,Bcl-2和Bcl-xl mRNA及蛋白表达下调,差异有统计学意义(P<0.05)。与空白对照组比较,SCF组卵母细胞凋亡率明显降低,Bax表达下调,Bcl-2和Bcl-xl表达上调,差异有统计学意义(P<0.05);与Cd组比较,SCF+Cd组卵母细胞凋亡率下降,Bax表达下调,Bcl-2和Bcl-xl表达上调,差异有统计学意义(P<0.05);与SCF+Cd组比较,SCF+Cd+ACK-2组卵母细胞凋亡率明显升高,Bax表达上调,Bcl-2和Bcl-xl表达下调,差异有统计学意义(P<0.05)。结论4 d龄新生大鼠卵巢体外培养镉暴露可引起卵母细胞的凋亡,同时抑制Scf及C-kit基因表达,外源性SCF可以改善镉暴露导致的卵母细胞的凋亡,其机制可能与SCF/c-kit信号通路有关。Objective To investigate the effect of stem cell factor on oocyte apoptosis induced by cadmium exposure in neonatal rats.Methods The ovaries of 4-day-old female SD rats were taken under sterile conditions and cultured in vitro and exposed to cadmium(CdCl_2)at doses of 0,0.5,5,10,50μmol/L for 4 days.The treatment of SCF was added in cell culture media in the further experiment,with the following groups:blank control group,100 ng/ml SCF、10μmol/L Cd、10μmol/L Cd+100 ng/ml SCF、10μmol/L Cd+100 ng/ml SCF+2μg/ml ACK-2(c-kit neutralizing antibody),in order to determine the correlation between oocyte apoptosis and the expression of SCF/c-kit of the ovary after cadmium exposure.The ultrastructure of oocyte apoptosis was observed by transmission electron microscope and the apoptosis rate of oocyte was detected by TUNEL method.The mRNA and protein expression levels of Scf,C-kit,Bax,Bcl-2 and Bcl-xl genes in ovarian tissues were detected by real-time PCR and Western blot respectively.Results Under transmission electron microscope,the oocyte cells showed concentrated nucleus,increase in heterochromatin the apoptotic bodies and swelling and vacuolation of mitochondria in all exposed groups.Compared with the control group,the oocyte apoptosis rate was increased in 0.5,5 and 10μmol/L cadmium-exposed group(P<0.05).The mRNA expression levels of Scf and C-kit in 5-50μmol/L group were significantly decreased(P<0.05).The mRNA and protein expression levels of Bax in cadmium-exposed group were increased(P<0.05),however,the expressions of Bcl-2 and Bcl-xl were down-regulated(P<0.05).Compared with the blank control group,the apoptotic rate of oocytes in SCF group was significantly decreased,the expression of Bax was down-regulated,the expressions of Bcl-2 and Bcl-xl were up-regulated(P<0.05).Compared with the Cd group,the apoptotic rate of oocytes in SCF+Cd group was decreased,the expression of Bax was down-regulated,and the expressions of Bcl-2 and Bcl-xl were up-regulated(P<0.05).Compared with the SCF+Cd group,the apoptosis

关 键 词：镉 卵巢培养 卵母细胞 细胞凋亡 干细胞因子 

分 类 号：R994.6[医药卫生—毒理学] R114[医药卫生—药学]