枸杞多糖对硫酸铍致人胚肺成纤维细胞bcl-2、bax及c-fos表达的影响

Protective Effect of Lycium Barbarum Polysaccharides on the Expression of Bcl-2,Bax and C-fos Induced by Beryllium Sulfate in Human Embryonic Lung Fibroblasts

目的探讨硫酸铍(BeSO_4)对体外培养人胚肺成纤维细胞凋亡相关蛋白bcl-2、bax及原癌基因c-fos蛋白表达的影响,以及枸杞多糖(LBP)的干预作用。方法体外培养人胚肺成纤维细胞(MRC-5)细胞系,分为空白对照组,低、中、高剂量BeSO_4染毒组(1、10、100μmol·L^(-1)),均分别体外培养24h、48h、72h;LBP干预组(0.2 mg·mL^(-1) LBP+10μmol·L^(-1) BeSO_4)及LBP对照组(0.2 mg·mL^(-1) LBP),分别培养48h。采用Western blot检测MRC-5细胞bcl-2、bax、c-fos蛋白表达水平。结果染毒48、72h时,各剂量组bcl-2蛋白的表达及中、高剂量组bcl-2/bax比值与对照组相比增高(P<0.05或P<0.01),染毒48h时,中、高剂量组c-fos蛋白的表达与对照组相比增高(P<0.01),染毒72h时,bax各剂量组的表达与对照组相比降低(P<0.05或<0.01),且存在剂量效应关系(P<0.01);同一剂量组,bcl-2蛋白的表达在48h之内随染毒时间的延长而增加,bcl-2/bax比值与c-fos蛋白的表达在72h之内随染毒时间的延长而升高,同一中、高剂量组,染毒24h之后,bax表达量随染毒时间的延长而降低,且存在时间效应关系(P<0.01)。LBP干预48h,bcl-2、c-fos、bcl-2/bax的表达与BeSO_4染毒组相比降低(P<0.05或<0.01),但bax的表达上调无统计学意义。结论 bcl-2和bax参与BeSO_4致MRC-5细胞凋亡的调控,原癌基因c-fos被激活,LBP可调节BeSO_4对细胞凋亡相关蛋白及原癌基因表达的影响。

Objective To explore protective effect of lycium barbarum polysaccharides（LBP） on protein expression of apoptotic regulatory genes（bcl-2 and bax）and a proto-oncogene（c-fos）induced by beryllium sulfate（BeSO4）. Methods Human embryonic lung fibroblasts（MRC-5 cell line） cultured in vitro were randomly divided into control-,low-,medium-and high-dose groups of BeSO4（1,10,and 100μM）,respectively,and they treated with BeSO4 for 24 h,48 h,72 h,respectively. fibroblasts of both intervention group（0.2 mg·m L^-1LBP ＋ 10μM BeSO4）and intervention control group（0.2 mg·m L^-1LBP）were treated with BeSO4 for 48 h.Protein expression level of bcl-2,bax and c-fos protein were detected by Western Blot. Results With the increase of exposure time and exposure dose,the cell density and morphological changes were found,the cells of exposure group were interdigitated while the growth arrangements were disordered and some cells transformation was different. After LBP intervention,cells states showed some improvement. Infected 48 h and 72 h,the expression of bcl-2 protein in the different dose groups and bcl-2/bax in the medium and high dose groups were significantly higher than those in the control group（P〈0.05 or0.01）and the expression of c-fos protein in the medium dose group and high dose group were significantly higher than that in the control group（P〈0.01）.Infected 72 h,the expression of bax in different doses groups was significantly lower than that in the control group（P〈0.05 or 0.01）and there was a dose-effect relationship（P〈0.01）. In the same dose group,the expression of bcl-2 protein increased with the prolongation of exposure time at 48 th hour while,bcl-2/bax and the expression of c-fos protein increased with the prolongation of exposure time at 72 nd hour. After 24 h,the expression of bax in the same medium and high dose group decreased with the prolongation of exposure time and there was a time-effect relationship（P〈0.01）. There were significant differences of protein expression of bcl-2,c-fos and bcl-2/bax between LBP intervention group and 10 μM BeSO4 group（P〈0.05 or P〈0.01）,but not for the expression level of bax. Conclusion Bcl-2 and bax were involved in the regulation of apoptosis of MRC-5 cells induced by BeSO4,c-fos was activated. LBP can adjust the effect of beryllium sulfate on apoptosis and the expression of proto-oncogene.