Detection Of Deletion Mutation Of Mitochondria DNA (mtDNA) In Mice By Real-time Fluorescent Quantitative PCR And Effect Of KangXin Liquid

Objective:To explore the mechanism of KangXin liquid (a prescription for reinforcing Kidney and Spleen,removing blood stasis) in retarding aging by investigating the effect of KangXin liquid intake on the of peripheral blood mitochondrial DNA(mtDNA) in aged Balb/c mice by real-time fluorescent quantitative PCR.Methods:Two fragments,267bp and 470bp long,represent mtDNA 4236bp deletion and wild-type mtDNA respectively, were amplified from the peripheral blood DNA of an aged mouse.They were cloned into pGEM-T Easy vector,resulting in the preparation of two recombinant vectors, pGEM-T Easy/267 and pGEM-T Easy/470.Plasmid DNA of these two vector was used in serial dilutions (from 10-2～10-5) as standerds to the protocol of.The mtDNA of the young mice(6-8 weeks),middle aged mice (6-8 months) and aged Balb/c mice(12-14months)were isolated respectively and deletion mutation was measured by real-time fluorescent quantitative PCR.The aged mice were divided into two groups,control group given 0.9% nomal saline,and KangXin Liquid group given KangXin Liquid.After four months,the deletions of peripheral blood mtDNA were detected.Results:Real-time fluorescent quantitative PCR for detection of the deletion mutation of mtDNA in mice was eastablished.There was 4236bp mtDNA deletion in peripheral blood mitochondria of aged Balb/c mice,but the same mtDNA deletion was not detected in young and middle aged mice.Compared with age-controlled group,KangXin Liquid could reduce the mtDNA deletion in aged mice.Conclusion:Real-time fluorescent quantitative PCR is an effective method for the detection of deletion mutation of mtDNA in mice,being sensitive and specific. Deletion mutation could accumulate with age.Intake of KangXin Liquid could obviously inhibit the occurred in peripheral blood mtDNA deletions to retard aging in aged mice.