Research And Analysis Of PolG On The Multi-differentiation Potential Of Adipose-derived Mesenchymal Stem Cells

Objective: DNA polymerase γ(PolG)is a mitochondrial DNA polymerase,which plays a key role in maintaining the stability of mitochondrial function and the integrity of mitochondrial DNA(Mitochondria deoxyribonucleic acid,mt DNA).PolG gene mutations can cause the instability of the mitochondrial genome,thereby making mitochondrial dysfunction.PolG-deficient mice exhibit age-dependent accumulation of DNA mutations and premature aging,and as age increases,their subcutaneous fat decreases and is accompanied by bone Poor texture.Adipose-derived stem cells(ADSCs)have the ability to differentiate into osteoblasts,adipocytes and chondrocytes.The metabolic mode of ADSCs in the differentiation process will change from glycolysis to aerobic oxidation,which will be accompanied by the synthesis of mt DNA and the enhancement of mitochondrial function.This project extracts ADSCs from PolGdeficient mice and wild-type(Wild Type,WT)mice,and induces differentiation,compares their differentiation ability,and then explores whether mitochondrial DNA damage and dysfunction affected by PolG gene mutations affect ADSCs Differentiation potential.Methods: 1.Breeding and breeding PolG heterozygous mice(PolG+/-),and crossing to obtain PolG knockout(Conventional Knockout,KO)mice(PolG-/-)and WT mice(PolG+/+).2.Select the littermates of PolG-KO mice and WT mice to extract,culture and identify mouse ADSCs.3.The third-generation WT ADSCs and Polg G-KO ADSCs were subjected to osteogenic induction,using quantitative real-time polymerase chain reaction(q PCR),alizarin red staining,and Western blotting.,WB)to detect osteogenic differentiation ability to test whether there is a difference in osteogenic differentiation ability between WT ADSCs and PolG-KO ADSCs.4.Adipogenic induction of the two ADSCs,oil red O staining and WB detection of their adipogenic differentiation ability to test whether there is a difference in the adipogenic differentiation ability of PolG-KO ADSCs and WT ADSCs.5.The third-generation WT ADSCs(WTP3),the sixthgeneration WT ADSCs(WTP6)of replicative aging and the third-generation PolG-KO ADSCs(KOP3)are used for cellular senescence β-galactosidase(senescence-associatedβ-Gal,SA-β-Gal)staining,WB experiment to detect P53 protein,adipogenic and osteogenic differentiation experiments to detect whether the differentiation potential of PolG-KO ADSCs and natural aging ADSCs are different.6.The lactic acid content of the cell culture medium of WT ADSCs and PolG-KO ADSCs and the cell culture medium after adipogenic differentiation was determined,and the expression of PolG protein during the differentiation process was detected by q PCR.WB detected the oxidative respiration related proteins Acetyl-Co A(Acetyl-Co A)and manganese superoxide dismutase(Mn SOD)to verify the role of PolG in the differentiation process of ADSCs.Results: 1.Alizarin red staining after induction of differentiation and osteogenic-related protein expression results prove that PolG gene defects can reduce the osteogenic differentiation ability of ADSCs.2.The results of oil red O staining and adipogenesisrelated protein expression test proved that the defects of PolG gene can reduce the adipogenic differentiation ability of ADSCs.3.The results of SA-β-Gal staining and the increase of P53 protein expression levels indicate that PolG gene defects can cause ADSCs to age,and the osteogenic differentiation results show that PolG-KO ADSCs and replicatively senescent ADSCs have similar results of reduced osteogenic differentiation.However,the results of adipogenic induction and differentiation showed that the adipogenic differentiation ability of PolG-KO ADSCs was reduced,and the adipogenic differentiation ability of replicatively senescent ADSCs was not significantly different from that of normal ADSCs.4.The results of the determination of the lactic acid content of the culture medium and the level of oxidative phosphorylation-related protein can infer that the PolG gene defect will inhibit the transformation of metabolic mode during the differentiation of ADSCs.Conclusion: PolG gene defects can lead to a decrease in the ability of ADSCs to differentiate.The reason may be due to the aging of ADSCs due to PolG gene defects and the inhibition of the transformation of metabolic methods during their differentiation.