Gadd45β和Gadd45γ在亚砷酸钠所致的MIHA细胞周期改变中的作用  

Role of Gadd45βand Gadd45γin sodium arsenite-induced alterations of cell cycle in MIHA cell

作　　者：刘诗文 王文静 孙宝飞[1] 张爱华[1] 魏绍峰[1] LIU Shi-wen;WANG Wen-jing;SUN Bao-fei;ZHANG Ai-hua;WEI Shao-feng(School of Public Health,the Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵州贵阳550025

出　　处：《现代预防医学》2022年第12期2254-2261,共8页Modern Preventive Medicine

基　　金：国家自然科学基金(81760572、81430077);贵州省教育厅基金(黔教科研发[2017]85号)。

摘　　要：目的探索Gadd45β和Gadd45γ在亚砷酸钠所致的MIHA细胞周期改变中的作用,为砷中毒的人群防治提供依据。方法收集贵州省兴仁县雨樟镇交乐病区砷中毒人群以及格沙屯正常人群分别作为砷中毒组和对照组,实时荧光定量PCR检测各组人群Gadd45β和Gadd45γ基因mRNA表达水平。同时,分别以不同剂量亚砷酸钠、不同时间处理MIHA细胞,流式细胞术测定细胞周期改变情况,实时荧光定量PCR和免疫印迹法分别检测Gadd45β和Gadd45γ基因m RNA表达水平和蛋白表达情况。在上述实验基础上,针对Gadd45β和Gadd45γ基因分别设计的siRNA作用于染砷的MIHA细胞,反向验证Gadd45β和Gadd45γ基因在砷致细胞周期改变过程中的影响。统计学分析采用独立样本t检验比较两组间差异,采用单因素方差比较多组间差异。结果人群实验研究发现,与对照组相比,砷中毒患者Gadd45β和Gadd45γ基因m RNA表达水平升高(t=2.576,P=0.011;t=2.312,P=0.022);MIHA细胞中,随亚砷酸钠染毒剂量、染毒时间增加,细胞G2/M期比例明显升高(F剂量=340.136,P<0.001;F_(时间)=49.194,P<0.001);在相对较低亚砷酸钠浓度(低于20μmol/L)、一定时间内(低于48 h)Gadd45β和Gadd45γ基因m RNA和蛋白表达水平随染砷剂量、染砷时间升高而升高,超过该浓度范围和作用时间后,出现表达下降的现象(Gadd45β:F_(剂量-蛋白)=37.568,P<0.001;F_(剂量-mRNA)=9.771,P<0.001;F_(时间-蛋白)=61.144,P<0.001;F_(时间-mRNA)=46.366,P=0.001;Gadd45γ:F_(剂量-蛋白)=12.989,P=0.001;F_(剂量-mRNA)=23.613,P<0.001;F_(时间-蛋白)=27.425,P<0.001;F_(时间-mRNA)=37.969,P<0.001)。转染si RNA分别下调Gadd45β和Gadd45γ的表达后,细胞周期都出现G2/M期比例下调(t=3.053,P=0.038;t=14.47,P<0.001)。结论砷致Gadd45β和Gadd45γ基因表达水平升高在其诱导MIHA细胞出现G2/M期阻滞过程中发挥重要作用。Objective To explore the role of Gadd45βand Gadd45γin MIHA cell cycle changes caused by sodium arsenite,to provide a basis for the prevention and control of arsenic poisoning population.Methods Arsenic poisoning population in Jiaoluo Ward,Yuzhang Town,Xingren County,Guizhou Province and normal population in Geshatun were collected as arsenic poisoning group and control group,respectively.The mRNA expression levels of Gadd45βand Gadd45γgenes were detected with qRT-PCR.At the same time,MIHA cells were treated with different doses of sodium arsenite or at different times,and the cell cycle changes were determined by flow cytometry,respectively,and the expression levels of mRNA and protein of the Gadd45βand Gadd45γgenes were measured by qRT-PCR and Western-blot.Based on the above experiments,siRNA designed for the Gadd45βand Gadd45γgenes,respectively,acts on arsenic-stained MIHA cells for reverse validation of the effects of the Gadd45βand Gadd45γgenes during arsenic-induced cell cycle changes.Statistical analyses used independent sample t-test to compare differences between two groups and one-way variance was used to compare differences between multiple groups.Results Compared with the control group,higher mRNA expression levels of Gadd45βand Gadd45γgenes were found in arsenic poisoning patients(t=2.576,P=0.011;t=2.312,P=0.022).In the MIHA cells,with the infection dose of NaAsO2and the infection time increased,the proportion of cell G2/M phase increased significantly(Fdose=340.136,P<0.001;Ftime=49.194,P<0.001).The mRNA and protein expression levels of Gadd45βand creased with increasing arsenic dose and time of arsenic staining at a relatively low sodium arsenite concentration(below 20μmol/L),for a certain time(below 48h),while the expression of Gadd45βand Gadd45γdecreased beyond this concentration range and the time of action(Gadd45β:F_(dose-protein)=37.568,P<0.001;F_(dose-m RNA)=9.771,P<0.001;F_(time-protein)=61.144,P<0.001;F_(time-mRNA)=46.366,P=0.001;Gadd45γ:F_(dose-protein)=12.989,P=0.001;F_(do

关 键 词：砷 GADD45Β Gadd45γ 细胞周期 MIHA细胞 

分 类 号：R599.1[医药卫生—内科学]