过量氟暴露对体内外星形胶质细胞及其胶质纤维酸性蛋白表达的影响  

Effects of excessive fluoride exposure on astrocytes and the expression of glial fibrillary acidic protein in vitro and in vivo

作　　者：傅晓丽 周箫 薄莉 李彬 何冬玲 官志忠 刘艳洁[1] Fu Xiaoli;Zhou Xiao;Bo Li;Li Bin;He Dongling;Guan Zhizhong;Liu Yanjie(Department of Pathology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Guizhou Key Laboratory of Molecular Biology,Key Laboratory of Molecular Biology,Guizhou Medical University,Key Laboratory of Endemic and Minority Diseases,Ministry of Education,Guiyang 550004,China)

机构地区：[1]贵州医科大学附属医院病理科,贵阳550004 [2]贵州省分子生物学重点实验室贵州医科大学分子生物学重点实验室地方病与少数民族疾病教育部重点实验室,贵阳550004

出　　处：《中华地方病学杂志》2022年第9期722-728,共7页Chinese Journal of Endemiology

基　　金：国家自然科学基金(81960572);国家自然科学基金委员会-贵州省人民政府联合基金子项目(U1812403);贵州省教育厅开放课题基金(黔教合KY字[2018]480)。

摘　　要：目的探讨过量氟暴露对体内外星形胶质细胞及其胶质纤维酸性蛋白(GFAP)表达的影响。方法(1)体内实验:选择24只SPF级SD大鼠,雌雄各半,按性别、体重采用随机数字表法分为对照组和染氟组,每组12只,分别饮用自来水配制的<1 mg/L和50 mg/L氟化钠溶液,饲养6个月。实验结束后采用免疫荧光法、免疫组织化学法和蛋白质免疫印迹法检测大鼠脑组织GFAP蛋白表达水平。(2)体外实验:分离成年(6月龄)大鼠大脑皮质星形胶质细胞进行原代培养(4 mmol/L氟化钠溶液培养24 h),采用免疫荧光法鉴定星形胶质细胞,实时荧光定量PCR法和蛋白质免疫印迹法分别检测GFAP mRNA及蛋白表达水平,流式细胞术检测星形胶质细胞凋亡情况及钙离子含量。结果(1)体内实验:免疫荧光法、免疫组织化学法、蛋白质免疫印迹法检测结果均显示,染氟组大鼠脑组织GFAP蛋白表达水平高于对照组(0.440±0.200比0.250±0.120,t=-5.93,P=0.027;0.270±0.020比0.240±0.050,t=-4.87,P=0.040;1.017±0.001比0.486±0.006,t=-52.48,P=0.001)。(2)体外实验:免疫荧光法鉴定结果显示,GFAP阳性,原代培养细胞鉴定为星形胶质细胞;实时荧光定量PCR法检测结果显示,染氟组GFAP mRNA表达水平高于对照组(2.780±0.120比0.134±0.005,t=-37.84,P=0.001);蛋白质免疫印迹法检测结果显示,染氟组GFAP蛋白表达水平高于对照组(2.76±0.10比1.38±0.05,t=-20.44,P=0.002);流式细胞术检测结果显示,染氟组细胞凋亡率高于对照组(%:55.0±1.0比3.5±0.6,t=-10.28,P=0.009),钙离子含量低于对照组(%:54±9比72±13,t=4.64,P=0.043)。结论过量氟暴露可造成体内外星形胶质细胞GFAP表达增加,细胞凋亡增多,并影响钙信号转导通路。Objective To investigate the effects of excessive fluoride exposure on astrocytes and the expression of glial fibrillary acidic protein(GFAP),in vitro and in vivo.Methods(1)In vivo experiment:24 SPF SD rats,half male and half female,were randomly divided into control and fluoride exposed groups according to sex and body weight,12 rats in each group.Rats were fed with<1 mg/L and 50 mg/L sodium fluoride solution prepared by tap water for 6 months,respectively.The expression levels of GFAP protein in rat brain tissue were measured by immunofluorescence,immunohistochemistry and Western blotting.(2)In vitro experiment:adult(6-month-old)rat cortical astrocytes were extracted and cultured in primary culture(4 mmol/L sodium fluoride solution for 24 h),and the astrocytes were identified by immunofluorescence,and GFAP mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting,respectively,and astrocytes apoptosis and calcium ion content were detected by flow cytometry.Results(1)In vivo experiment:the results of immunofluorescence,immunohistochemistry and Western blotting showed that the GFAP protein expression level in brain tissue of rats exposed to fluoride was higher than that of control group(0.440±0.200 vs 0.250±0.120,t=-5.93,P=0.027;0.270±0.020 vs 0.240±0.050,t=-4.87,P=0.040;1.017±0.001 vs 0.486±0.006,t=-52.48,P=0.001).(2)In vitro experiment:GFAP positive cells were identified as astrocytes by immunofluorescence;GFAP mRNA expression level was higher in fluoride exposed group than that of control group by real-time fluorescence quantitative PCR(2.780±0.120 vs 0.134±0.005,t=-37.84,P=0.001).The Western blotting results showed that the GFAP protein expression level was higher in fluoride exposed group than that of control group(2.76±0.10 vs 1.38±0.05,t=-20.44,P=0.002).Flow cytometry results showed that the apoptosis rate of astrocytes was higher in fluoride exposed group than that of control group(%:55.0±1.0 vs 3.5±0.6,t=-10.28,P=0.009)and the calcium ion

关 键 词：氟化物中毒 胶质纤维酸性蛋白 星形胶质细胞 原代细胞培养 

分 类 号：R599.1[医药卫生—内科学]