黄芪甲甙对衰老HELF细胞端粒酶活性、细胞周期因子及klotho基因调控作用的实验研究
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摘要
目的:探讨黄芪甲甙(Astr)对衰老人胚肺成纤维细胞(HELF)的影响,通过HELF细胞活力、SA-β-gal活性、端粒酶活性和cyclinD1、cyclinE、CDK4、p16、p21、klotho mRNA表达的变化,说明黄芪甲甙对衰老HELF细胞作用的机制。
方法:采用体外培养复制性衰老HELF细胞为研究对象,将细胞分成四组:青年组(35代),衰老组、黄芪甲甙组和对照组。HELF细胞进行体外常规培养,细胞从50代按照1μg黄芪甲甙/ml培养基开始加入黄芪甲甙,培养至53代,观察细胞形态,采用MTT法检测细胞活力,Dimiri氏法检测β-半乳糖苷酶活性,TRAP-PCR银染法检测端粒酶活性;RT-PCR法检测cyclinD1、cyclinE、CDK4、p16、p21、klotho mRNA表达。
结果:与青年组比较:衰老组细胞活力明显降低,CDK4、klotho mRNA表达均明显降低(p<0.05),β-半乳糖苷酶活性,cyclinD1、cyclinE、p16、p21 mRNA表达均升高(p<0.05)。与衰老组和对照组相比:黄芪甲甙组细胞β-半乳糖苷酶活性明显降低,cyclinD1、cyclinE、p16、p21 mRNA的表达水平明显降低(p<0.05),而细胞活力、端粒酶活性和CDK4、klotho mRNA的表达明显提高(p<0.05)。
结论:黄芪甲甙可发挥抗HELF细胞衰老作用,使衰老HELF细胞活力和端粒酶活性提高,β-半乳糖苷酶活性降低。其作用可通过上调CDK4、klotho基因mRNA的表达,从而抑制p21通路,降低cyclinD1、cyclinE、p16、p21 mRNA的表达水平,而实现其抗HELF细胞衰老的作用。
Objective:To probe into the effects of astragaloside on the activity of SA-β-gal and telomerase,and the mRNA expression of cyclinD1,cyclinE,CDK4,p16,p21 and klotho,an anti-aging gene,on human embryonic lung fibroblast.
Methods:We dividd HELF into four groups in this study,they are group of young,group of astragaloside,group of control and group of aging.In vitro human embryonic lung fibroblast,add the astragaloside when the cell grow up to 50 generations in test group.We observe the cell shape with the inverted Microscopy,and measured the cell viability by MTT method,the activity ofβ-Galactosidase by Dimiri’s method,the activity of telomerase TRAP-PCR by Silver staining method,the mRNA expression of cyclinD1,cyclinE,CDK4,p16,p21 and klotho by RT-PCR method.
Results:Compare with the youth group,the aging group cell viability and telomerase activity descent,β-galactosidase activity and the mRNA expression of cyclinD1,cyclinE,p16 and p21 goes up(p<0.05),the expression of klotho and CDK4 was fall down (p<0.05);compare with aging group,the activity ofβ-Galactosidase and the expressiones of cyclinD1,cyclinE,p16 and p21 were reduced,which effected by astragaloside.The cell viability,the activity of telomerase,the mRNA expression of CDK4,and klotho raise up.
Conclusions:Astragaloside react the effect of anti-aging by enhanced to the activity of telomerase and mRNA expression of cyclinD1,cyclinE,CDK4,p16,p21 and klotho.
方法:采用体外培养复制性衰老HELF细胞为研究对象,将细胞分成四组:青年组(35代),衰老组、黄芪甲甙组和对照组。HELF细胞进行体外常规培养,细胞从50代按照1μg黄芪甲甙/ml培养基开始加入黄芪甲甙,培养至53代,观察细胞形态,采用MTT法检测细胞活力,Dimiri氏法检测β-半乳糖苷酶活性,TRAP-PCR银染法检测端粒酶活性;RT-PCR法检测cyclinD1、cyclinE、CDK4、p16、p21、klotho mRNA表达。
结果:与青年组比较:衰老组细胞活力明显降低,CDK4、klotho mRNA表达均明显降低(p<0.05),β-半乳糖苷酶活性,cyclinD1、cyclinE、p16、p21 mRNA表达均升高(p<0.05)。与衰老组和对照组相比:黄芪甲甙组细胞β-半乳糖苷酶活性明显降低,cyclinD1、cyclinE、p16、p21 mRNA的表达水平明显降低(p<0.05),而细胞活力、端粒酶活性和CDK4、klotho mRNA的表达明显提高(p<0.05)。
结论:黄芪甲甙可发挥抗HELF细胞衰老作用,使衰老HELF细胞活力和端粒酶活性提高,β-半乳糖苷酶活性降低。其作用可通过上调CDK4、klotho基因mRNA的表达,从而抑制p21通路,降低cyclinD1、cyclinE、p16、p21 mRNA的表达水平,而实现其抗HELF细胞衰老的作用。
Objective:To probe into the effects of astragaloside on the activity of SA-β-gal and telomerase,and the mRNA expression of cyclinD1,cyclinE,CDK4,p16,p21 and klotho,an anti-aging gene,on human embryonic lung fibroblast.
Methods:We dividd HELF into four groups in this study,they are group of young,group of astragaloside,group of control and group of aging.In vitro human embryonic lung fibroblast,add the astragaloside when the cell grow up to 50 generations in test group.We observe the cell shape with the inverted Microscopy,and measured the cell viability by MTT method,the activity ofβ-Galactosidase by Dimiri’s method,the activity of telomerase TRAP-PCR by Silver staining method,the mRNA expression of cyclinD1,cyclinE,CDK4,p16,p21 and klotho by RT-PCR method.
Results:Compare with the youth group,the aging group cell viability and telomerase activity descent,β-galactosidase activity and the mRNA expression of cyclinD1,cyclinE,p16 and p21 goes up(p<0.05),the expression of klotho and CDK4 was fall down (p<0.05);compare with aging group,the activity ofβ-Galactosidase and the expressiones of cyclinD1,cyclinE,p16 and p21 were reduced,which effected by astragaloside.The cell viability,the activity of telomerase,the mRNA expression of CDK4,and klotho raise up.
Conclusions:Astragaloside react the effect of anti-aging by enhanced to the activity of telomerase and mRNA expression of cyclinD1,cyclinE,CDK4,p16,p21 and klotho.
引文
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