Effect Of Lentivirus Vector-mediated Apelin Gene Transfection On Glucose Uptake In Insulin Resistance 3T3-L1 Adipocytes

Objective: Observe the effect of lentiviral vector-mediated Apelin gene transfection on glucose uptake in insulin resistant 3T3-L1 adipocytes.Methods:(1)3T3-L1 preadipocytes were co-administered with 0.5mM 3-isobutyl-1-methylxanthine,1 μM dexamethasone and 10 μg/ml insulin,and induced into mature adipocytes,oil red "O" identification of staining;(2)TNF-alpha stimulation induced the establishment of 3T3-L1 adipocytes insulin resistance model,and glucose uptake kits were used for detection and identification;(3)A lentiviral vector carrying the Apelin gene was constructed by subcloning the gene,and the lentiviral fluid was packaged with HEK293 T cells,and the concentrated virus solution was used to infect the insulin-resistant 3T3-L1 adipocytes,(1)RNA was extracted from the cells,using RT-PCR to confirm the expression of Apelin gene.(2)Extracted the intracellular protein,and Western Blot experiment was used to verify the expression of Apelin protein.(3)The glucose uptake kit was used to detect the change of glucose uptake.Results:(1)Oil red “O” staining results confirmed that more than 80% of 3T3-L1 preadipocytes successfully induced mature adipocytes;(2)Compared with the control group,TNF-α reduced the insulin uptake of insulin-stimulated 3T3-L1 adipocytes by 32.7%.The difference was statistically significant(P<0.05);(3)Apelin gene was transfected into insulin-resistant 3T3-L1 adipocytes,(1)RT-PCR confirmed that Apelin gene fragment was successfully amplified by transfecting Apelin gene.(2)Western Blot experiment confirmed that there were obvious protein bands around 8Kd in transfected Apelin gene cells,but no obvious protein bands appeared in the untransfected control group.(3)Compared with the untreated control group,the glucose uptake test confirmed that the transfection of Apelin gene significantly increased the glucose uptake by 42.2%.The difference was statistically significant(P<0.01).Conclusion: Lentiviral vector-mediated Apelin gene successfully transfected insulin-resistant 3T3-L1 adipocytes,which significantly increased glucose uptake,thus improving insulin resistance.