氟化钠对L02人正常肝细胞衰老及相关蛋白表达影响的实验研究  

Effect of sodium fluoride on senescence and related protein expression in L02 normal hepatocytes

作　　者：周莉[1] 毛春燕 李旭峰 倪鹏飞 齐晓岚 ZHOU Li;MAO Chun-yan;LI Xu-feng;NI Peng-fei;QI Xiao-lan(Department of Health Management,Guiyang Vocational College of Nursing,Guiyang,Guizhou 550081,China)

机构地区：[1]贵阳护理职业学院卫生管理系,贵州550081 [2]贵州医科大学地方病与少数民族疾病教育部重点实验室

出　　处：《现代预防医学》2020年第23期4331-4334,4365,共5页Modern Preventive Medicine

基　　金：贵阳市科技计划项目(筑科合同[2017] 30-3);国家自然科学基金-贵州省人民政府联合基金(U1812403)。

摘　　要：目的研究氟化钠(NaF)是否会诱导人肝细胞L02衰老及衰老相关标志蛋白水平的改变。方法不同浓度(0、1、2、3、4、5、6、7和8 mmol/L) NaF处理L02人正常肝细胞,CCK-8法检测细胞存活率的变化以筛选NaF的作用浓度;衰老标志物β-半乳糖苷酶(β-galactosidase,β-Gal)染色检测衰老阳性细胞;实时荧光定量PCR (Real-time PCR)和蛋白免疫印迹(Western Blot)测定细胞衰老标志蛋白p16,p21 mRNA和蛋白的表达水平;流式细胞术分析细胞周期的变化情况。结果 CCK-8结果显示,NaF浓度为1、4、7 mmol/L时L02细胞的存活率(87.38±0.93、66.72±2.81、52.17±2.04)均低于对照组[(100.00±0.00),均P<0.01],且每组之间的细胞存活率差异存在统计学意义(均P<0.01),因此NaF选用1、4、7 mmol/L处理L02细胞作为低氟组、中氟组、高氟组开展研究;β-半乳糖苷酶染色结果显示,低、中、高氟组中细胞衰老率(23.15±0.23、36.06±0.40、65.36±0.82)均高于正常组[(3.27±0.27),P<0.01];Real-time PCR结果显示,p16、p21 mRNA在低、中、高氟组中表达(2.29±0.19、3.44±0.30、5.25±0.32;1.88±0.22、3.22±0.24、7.33±0.30)表达均高于正常对照组[(1.00±0.00;1.00±0.00),均P<0.01]。低、中、高氟组中p16蛋白水平(93.68±3.40、116.25±7.30、122.31±3.00)均高于对照组[(78.07±4.17),P<0.05,P<0.01,P<0.01),低、中、高氟组中p21蛋白水平(87.61±5.22、121.62±4.27、147.95±7.81)均高于对照组[(61.32±4.56),均P<0.01)];流式结果显示,低、中、高氟组L02细胞周期均停滞在G2期,与对照组相比,差异具有统计学意义(P<0.01)。结论 NaF处理的人肝细胞L02中细胞衰老率及衰老标志蛋白p16、p21的mRNA和蛋白表达升高,这可能提示了NaF可导致L02细胞衰老,进一步说明细胞衰老在氟化物诱导的肝损伤中可能具有重要作用。Objective To investigate whether different concentrations of NaF can induce cell senescence of human hepatocytes L02 cells and elevate the level of senescence related proteins.Methods L02 cells were treated with NaF at different concentrations(0,1,2,3,4,5,6,7 and 8 mmol/L).Cck-8 was used to detect the change of cell survival rate.Senescence markerβ-Gal staining was used to detect senescence positive cells.Real-time PCR and Western Blot were used to determine the mRNA and protein levels of senescence markers p16,p21.Flow cytometry was used to analyse the changes of cell cycle.Results CCK-8 results showed that the survival rate of L02 cells(87.38±0.93,66.72±2.81 and 52.17±2.04)at NaF concentration of 1,4 and 7 mmol/L was lower than that of the control group[(100.00±0.00),all P<0.01),and there was a significant difference in cell survival rate between each group(all P<0.01).Therefore,NaF treated L02 cells with 1,4 and 7 mmol/L as the low-fluorine,medium-fluorine and high-fluorine groups.The results ofβ-galactosidase staining showed that the cell senescence rate(23.15±0.23,36.06±0.40,65.36±0.82)in the low,medium and high fluoride groups was higher than that in the normal group[(3.27±0.27),P<0.01].Real-time PCR results showed that the expression of p16 and p21 mRNA in the low,medium and high fluoride groups(2.29±0.19,3.44±0.30,5.25±0.32;1.88±0.22,3.22±0.24,7.33±0.30)were all higher than Normal control group[(1.00±0.00;1.00±0.00),all P<0.01].The levels of p16 protein(93.68±3.40,116.25±7.30,122.31±3.00)in the low,medium,and high fluoride groups were higher than those in the control group[(78.07±4.17),P<0.05,P<0.01,P<0.01).The levels of p21 protein(87.61±5.22,121.62±4.27,147.95±7.81)in the low,medium,and high fluoride groups were all higher than those in the control group[(61.32±4.56),all P<0.01)].The flow cytometry results showed that the L02 cell cycle of the low,medium,and high fluoride groups were all arrested in G2 phase,and the difference was statistically significant compared with the con

关 键 词：氟 细胞衰老 P16 P21 肝细胞L02 

分 类 号：R114[医药卫生—卫生毒理学]