松花粉对衰老成纤维细胞线粒体DNA缺失突变的影响

摘要

目的 通过研究松花粉对衰老细胞线粒体(mt) DNA4977缺失突变的影响,探讨松花粉抗衰老的作用机制.方法 将细胞分为青年组、衰老细胞组、含240 mg/dl松花粉的衰老细胞处理组,三组细胞分别用不同培养基培养后,抽提线粒体DNA,进行PCR检测mtDNA4977缺失突变,以线粒体DNA中保守序列PCR扩增结果作为内参,比较各组mtDNA4977缺失突变在总线粒体DNA中的比例；同时对各组细胞进行β-半乳糖苷酶染色；并测定各组细胞超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量.结果 松花粉能够改善衰老成纤维细胞的衰老变化,并能降低衰老细胞mtDNA4977的缺失突变,提高细胞SOD活性及降低其MDA含量(P＜0.05).结论 松花粉可能通过减轻成纤维细胞的氧化损伤,从而保护细胞mtDNA延缓成纤维细胞的衰老.%Objective To detect the influence of pine pollen on the mitochondria DNA (mtDNA) deletions of aging cells and investigate the antidotal mechanism of pine pollen. Methods Cells were divided into three groups: the young cells were determined as group A and fed by normal DMEM, aging cells were determined as group B and fed by normal DMEM, aging cells in the research group fed by DMEM containing 240 mg/dl pine pollen and named as group C. The intracellular total mtDNA was extracted from the cells in different groups. Using a conservative fragment in the mtDNA as an internal standard, the relative proportion of mtDNA4977 in the total mtDNA was determined by a competitive polymerase chain reaction ( PCR) method,the positive rate of SA-β-galactosidase stain was determined by immu-nohistochemistry. Relative level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in cells were measured. Results After the treatment of pine pollen, positive rate of SA-β-gal was significantly decreased ( P < 0. 05 ) , pine pollen could down regulate the proportion of the mtDNA4977 deletion in the total mtDNA of the aging cells. Pine pollen could decrease MDA volume and increase the activity of SOD significantly ( P < 0. 05 ). Conclusions Pine pollen can reduce oxidative damage of mtDNA and protect mtDNA. Accordingly it provides the possible mechanism of anti-aging effect of Pine pollen from the molecular level.