The Roles Of Caveolin-1 In The Growth Characteristics Of 3T3-L1 Cells And In The Insulin Signal Pathway

Objective: To study the the effects of Caveolin-1 on the growth characteristicsof 3T3-L1 cells and to unravel the role of Caveolin-1 in the insulin signal pathway.Methods: The vector with the wild CAV1 gene, and the CAVRNAi vector weretansfected into the 3T3-L1 cells respectively.The stably transfected cell lines wereselected with the corresponding bioepiderm resistance.The expression of the CAV1protein was tested by western blot; the cell cycle distribution was tested by flowcytometry; the expression and location of the insulin pathway associated protein IR,CAV1, GLUT4 were tested by immunofluorescence. Western blot was used to testthe expression and location of the proteins of CAV1, GLUT4 and IR.Reults: The expression levels of CAV1 in 3T3-L1 cells transfected withpcDNA3.1/NT-GFP-CAV1 was higher than that in other groups(P??0.05); theexpression level of CAV1 in cells with CAV1 Lentiviral RNAi was lower than that inthe other groups??P <0.05??,and no significant differences were observed between theGroup 3T3 and Group GFP(P >0.05??.The results of flow cytometer showed that in Group CAV1-GFP the proportionof the cells in the period of G0/G1 increased, while the cells in G2/M decreased??P<0.05??; in Group RNAi the proportion of the cells in G0/G1 decreased, whilethe cells in the period of G2/M increased??P<0.05??; it has no statistic differencebetween the Group normal TT cells and Group blank vector.The results of testing the expression of Caveolin-1 and IR with fluorescencemicroscope showed that CAV1 and IR in the cells of Group CAV1, Group3T3 andGroup GFP released red fluorescent lights, while the CAV1 and IR in the cells ofGroup RNAi released green fluorescent lights, and they were distributed sporadicly inthe kytoplasm. In conclusion, for the fluorescentsignals, Group CAV1 was thestrongest, Group 3T3 and Group GFP were the second, and Group RNAi was theweakest. Except for that of Group RNAi, the fluorescence intensity of all the Groups increased with the increase of RI, and it was the strongest when the IR was added atthe concentration of 300MU/L. The tendency above was most significant in GroupCAV1 and the least in Group GFP and Group 3T3, while it was not significant inGroup RNAi.The proteins were collected at 6 hours after treating the 6 groups of cells withthe RI at the concentration of 0MU/L and 300MU/L. Western blot results showed thatthe expression level of CAV1 protein in the cells of Group CAV1, Group3T3 andGroup GFP were higher than that in Group RI-free??P<0.05??, and the difference wasnot significant in Group RNAi??P??0.05??. The expression level of CAV1 protein wasthe highest in Group CAV1, second in Group GFP and Group 3T3, and the lowest inGroup RNAi.Conclusion: 1. The 3T3-L1 cell lines stably expressing complete CAV1 gene wasestablished respectively. The 3T3-L1 cell line with CAV1 gene silencing wasdevoleped .2. Excessively stably expression of promotes CAV1 the growth of 3T3-L1cells.3. Insulin can induce the expression of the protein of CAV, IR and GLUT4 in aconcentration dependent manner and the tendency is significant in the 3T3-L1 cellsexcessive CAV1 gene expression, while it doesn't exist in the cells of with GroupCAV1 gene silence.