Preliminary Study On Brown Differentiation Of Human Adipose-derived Mesenchymal Stem Cell Induced By Adenovirus Type 36

Objective: To investigate the role of adenovirus type 36(Ad36)in the induction of differentiation of human adipose-derived mesenchymal stem cells into brown adipocytes.Methods:(1)hADSCs were induced by cocktail and Ad36 respectively,and stained with oil red O.The cells were observed for adipogenesis at 2,4,6,and 8 days after induction.(2)Cells of the cocktail induction group and Ad36 induction group were collected on days 0,2,4,6,and 8 respectively.The RT-qPCR method was used to detect the LncRNA ROR in the two groups,and brown adipocyte differentiation marker genes Cidea,Dio2,Fgf21,Ucp1,Prdm16,mitochondrial respiratory chain and oxidative phosphorylation-related genes Cox5 b,Atp5o,Atp6,Nd2,Leptin,etc.mRNA expression levels;Western-blotting detection of the above two genes in the protein expression levels.(3)In the process of hADSC differentiation induced by Ad36,LncRNA ROR was knocked down by siRNA,and cells from knock-down group and control group were harvested on days 0,1,2,3,and 4 respectively.RT-qPCR and Western-blotting methods were used to detect the above-mentioned brown adipocyte differentiation marker genes,mitochondrial respiratory chain and oxidative phosphorylation-related enzymes mRNA and protein expression levels.(4)Using transmission electron microscopy to observe and calculate the changes and differences in the number of mitochondria during induction of differentiation.Results:(1)The results of oil red O staining showed that in the differentiation process of hADSC,lipid droplets in the adipocytes of the cocktail-induced group were larger,while those in the Ad36-induced group were granular smaller lipid droplets.(2)RT-qPCR and Western-blotting results showed that in the differentiation process of hADSC,LncRNA ROR,Cidea,Dio2,Fgf21,Ucp1,Prdm16,Cog5 b,Atp5o,Atp6,Nd2 in the Ad36-induced group compared with the cocktail-induced group.The mRNA and protein expression levels were significantly increased,and the difference was statistically significant(P<0.01 or P<0.05).The mRNA and protein expression levels of Leptin were significantly decreased,and the difference was statistically significant(P<0.01).(3)The mRNA and protein expression levels of Cidea,Dio2,Fgf21,Ucp1,Prdm16,Cox5 b,Atp5o,Atp6,and Nd2 were significantly decreased after knockdown of the LncRNA ROR by Ad36-induced hADSC differentiation.The difference was statistically significant(P<0.01 or P<0.05).(4)Transmission electron microscopy showed that during adipocyte differentiation,the number of mitochondria in the Ad36-induced group was significantly higher than that in the cocktail-induced group,and the difference was statistically significant.Conclusion: Ad36 may accelerate mitochondrial oxidative phosphorylation and energy expenditure by up-regulating LncRNA ROR,which in turn promotes the differentiation of hADSC into brown adipocytes.