Study Of The Role Of Wnt Signaling Molecules In Fat Metabolism And Its Protein Nature

Wnt proteins were secreted from cells and recognized the memberreceptor Frizzled(Fzd),which actived the target cells.The signalingmolecules of wnt family regulated numerous processes in animaldevelopment and had been increasingly implicated in adipogenesis.Thisstudy used RNA interference(RNAi) to detect the function of Wnt5band Fzd4 during the adipogenesis differentiation processes.3T3-L1 cell differented into fat cells and was used in the study ofadipocyte differentiation. We used RT-PCR to detect the express levelof wnt genes during 3T3-L1 cell differented process.The expressionlevel of Wnt5b was higher during adipogenesis differentiation thanpreadipocytes. Fzd4 was highly expressed during the adipogenesisdifferentiation process. So RNAi technique was used to silence theexpression of Wnt5b and Fzd4 in 3T3-L1 cells. Because thetransfecting efficiency of 3T3-L1 cells was low, we transfected theRNAi vectors and the genes into 293T cells that easier to betransfected.The expression level of this two genes were detected andthe efficient RNAi targets were selected, and so they were transfectedinto 3T3-L1 cells and the functions were observed.Specific RNAi target sequences were selected from Fzd4 gene andWnt5b gene.The 62bp oligonucleotide fragment was inserted into theretroviral vector which included the H1 promoter and destabilizedgreen florescence protein. The plasmids were transfected into 293Tcells and the expression level of gene was detected by Western blotting.The expression level of Fzd4 gene and Wnt5b gene were significantlyinhibited by RNAi vector. The efficient RNAi targets were selected andtransfected into Phenoix cells.The retrovirus was collected to infect the3T3-L1 cells,which were stained with Oil Red O stock solution.Thestained lipid droplets in the cell were extracted with isopropyl alcohol,and the absorbance was measured at 490 nm. We examined theexpressions of lipid droplets with Fzd4 RNAi targets in 3T3-L1adipocytes and found the lipid droplets were increased. The resultsindicated that Fzd4 inhibited adipogenesis in 3T3-L1 cells.We alsoexamined the influence of Wnt5b RNAi target to 3T3-L1 adipocytesand found that the expressions of lipid droplets were decreased.Theresults indicated that Wnt5b promoted adipogenesis in 3T3-L1 cells.We expressed the protein of Wnt5b and Wnt9a and acquired theantibody serum of Wnt5b.