双歧杆菌脂磷壁酸延缓细胞衰老的作用及其机制的初步探讨

发布时间：2018-10-10 10:05

【摘要】： 实验目的双歧杆菌是人和动物肠道内重要的生理菌,其表面分子脂磷壁酸(Lipoteichoic acid ,LTA)已被证实具有抗氧化的作用,能够有效延缓衰老,但其机制并不是很清楚。本研究用H2O2诱导人胚肺二倍体成纤维细胞WI-38出现“应激诱导早熟性老化”(stress-induced premature senescence,SIPS),观察双歧杆菌LTA是否具有对抗H2O2诱导细胞衰老的作用,以及WI-38细胞中p21、CDK2和cyclin E mRNA和蛋白表达水平的变化,初步探讨双歧杆菌LTA抗细胞衰老的细胞周期调控机制。实验方法 1.采用噻唑兰比色(MTT)法检测经双歧杆菌LTA处理前后,人胚肺二倍体成纤维细胞WI-38生存率影响的变化;用亚致死量的H2O2诱导细胞衰老,β-半乳糖苷酶细胞化学染色检测衰老细胞,计算其阳性百分率并检测经双歧杆菌LTA处理前后对β-半乳糖苷酶细胞化学染色阳性百分率的变化。 2.双歧杆菌LTA体外作用人胚肺二倍体成纤维细胞WI-38后,用RT-PCR检测衰老相关指标p21、cyclin E和CDK2的mRNA水平变化,Western blot分析p21、cyclin E和CDK2蛋白水平的变化。实验结果 1.经双歧杆菌LTA作用后,人胚肺二倍体成纤维细胞WI-38的生存率增高并且β-半乳糖苷酶细胞化学染色阳性率降低。 2.经双歧杆菌LTA作用后,WI-38细胞中与衰老相关的p21的mRNA及蛋白表达水平下降;cyclin E和CDK2的mRNA及蛋白表达水平升高。实验结论 1.双歧杆菌LTA能抑制H2O2对WI-38细胞氧化损伤作用,抑制H2O2对WI-38细胞诱导衰老的作用。 2.双歧杆菌LTA能明显下调WI-38细胞中p21的mRNA和蛋白质的表达;上调WI-38细胞中cyclin E和CDK2的mRNA及蛋白表达水平。
[Abstract]:Objective Bifidobacterium is an important physiological bacterium in human and animal intestinal tract. Its surface molecule (Lipoteichoic acid has been proved to have antioxidant effect and can effectively delay senescence, but its mechanism is not very clear. In this study, H2O2 was used to induce human embryonic lung diploid fibroblasts (WI-38) to appear "stress-induced premature aging" (stress-induced premature senescence,SIPS). Whether the bifidobacterium LTA could antagonize the senescence induced by H2O2 was observed. The changes of expression of p21 CDK2, cyclin E mRNA and protein in WI-38 cells were also studied to explore the cell cycle regulation mechanism of bifidobacterium LTA against cell senescence. Experimental method 1. The changes of WI-38 survival rate of human embryonic lung diploid fibroblasts treated with bifidobacterium LTA were detected by thiazolam colorimetric (MTT) assay, the senescence of cells was induced by sublethal H2O2, and the aging cells were detected by 尾 -galactosidase cytochemical staining. The positive percentage of 尾 -galactosidase was calculated and the changes of 尾 -galactosidase cytochemical staining were detected before and after treatment with Bifidobacterium LTA. 2. Bifidobacterium LTA was used to detect the mRNA levels of p21 cyclin E and CDK2 in human embryonic lung diploid fibroblasts treated with WI-38 in vitro. Western blot was used to analyze the changes of p21 cyclin E and CDK2 protein levels. Experimental results 1. After treated with Bifidobacterium LTA, the survival rate of human embryonic lung diploid fibroblasts increased and the positive rate of 尾 -galactosidase cytochemical staining decreased. 2. The expression level of mRNA and protein of senescence related p21 in WI-38 cells was decreased after treatment with bifidobacterium LTA. The expression of mRNA and protein of cyclin E and CDK2 was increased. Conclusion 1. Bifidobacterium LTA inhibited the oxidative damage of H2O2 to WI-38 cells and the senescence of WI-38 cells induced by H2O2. 2. Bifidobacterium LTA could significantly down-regulate the expression of p21 mRNA and protein in WI-38 cells, and up-regulate the mRNA and protein expression of cyclin E and CDK2 in WI-38 cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R371

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