The Construction Of SOCS3 Eukaryotic Expression Vector And Effection On Apoptosis Of The Adipose Cells

The member of SOCS (Suppressor of cytokine signaling) family proteins have similar structure, include the SH2 area, N area and about 40 amino acids from the C-terminal of SOCS box. They were initially thought to regulate the negative feedback regulator of JAK/STAT signaling pathway, and SOCS3 was confirmed of the negative feedback regulation signaling pathway in many studies such as Leptin (Leptin), Tumor Necrosis Factor-α(TNF-α), Interferon-γ(IFN-γ), Growth hormone (GH). In recent years, studies of SOCS3 have shown that it plays an important role on cell growth, differentiation and apoptosis. In this study we take mouse preadipocytes cells and 3T3-L1 cells as the research object, which was aimed to focus on SOCS3 adipocyte apoptosis. At the Begining, RNA was extracted from mouse adipose tissue as a template, by RT-PCR amplification, SOCS3 gene was cloned into pMD18-T, constructed pMD18-T-SOCS3 plasmid and took pMD18-T-SOCS3 as a template, SOCS3 gene was amplified and cloned to pEGFP-N1, constructed eukaryotic recombinant plasmid PEGFP-N1-SOCS3; and then a successful recombinant plasmid pEGFP-N1-SOCS3 was transfected to preadipocytes cells and mouse 3T3-L1 cells, by fluorescence microscope, we can observate GFP reporter gene expression, by RT-PCR we detected apoptosis-related genes Bax, c-myc, bcl-2, mcl-1, survivin etc. and JAK2 and STAT5 gene, by Western blotting we detected intracellular SOCS3 mRNA and protein expression, Our study is aimed at preliminary study about effection of SOCS3 on the fat cells apoptosis and on JAK2/STAT5 signaling pathway, the main results are as follows:1. By RT-PCR, we have cloned region of mouse SOCS3 gene with cds, in which cDNA full-length sequence is 678bp, Genbank accession number is No.NM₀07707.3. The amino acid sequence of SOCS3 was analysised by using SignalP 3.0 Server,Neural Networks (NN) and Hidden Markov Models (HMM) , result showed that the possibility of SOCS3 (cds) is not signal peptide, a non-secreted protein, and we used SWISS-MODEL to predict areas of SOCS3 cds protein 3D structure.2. Take RNA Extracted from mouse adipose tissue as a template, by RT-PCR we obtained SOCS3 cds cDNA, cloned into pMD18-T and pEGFP-N1 and built pMD18-T-SOCS3 and pEGFP-N1-SOCS3 plasmid, which was identified by restriction enzyme digestion and PCR positive clones, and the sequence was proved to insert pMD18-T-SOCS3 and pEGFP-N1 correctly, which comfirmed expression vector pEGFP- N1-SOCS3 was successfully constructed.3. With pEGFP-N1-SOCS3 was transfected into 3T3-L1 cells by ways of liposome with control group and liposome group. we found that liposome group and SOCS3 group can be observed with reporter gene GFP expression under a fluorescence microscope; by ways of RT-PCR and Western blotting, we abserved SOCS3 mRNA and protein expression was significantly increased (P<0.05) in the 3T3-L1 cells; Hoechst 33258 staining show that SOCS3 group, apoptosis was significantly; Bax and c-myc gene mRNA expression was significantly increased (P<0.05), bcl-2 and mcl-1 gene expression was significantly decreased (P<0.05). SOCS3 can promote the occurrence of apoptosis on 3T3-L1 cells.4. By ways of liposome transfection we took the pEGFP-N1-SOCS3 to mouse preadipocytes, we made the control group and liposome group and observed that liposome group and SOCS3 group that the report gene GFP was expressed under a fluorescence microscope; by ways of RT-PCR and Western blotting, we found that SOCS3 mRNA and protein expression in the preadipocytes cells was significantly increased (P<0.05); Bax, c-myc and survivin gene mRNA expression level was significantly higher (P<0.05), bcl-2, mcl-1 and NF-kB gene expression level was significantly lower (P<0.05). The results showed that SOCS3 can promote the apoptosis on mice preadipocytes.5. We took the pEGFP-N1-SOCS3 to mouse preadipocytes through liposome transfection, using the same method with No.4 tips. we tested gene expression of JAK2,STAT3, the result showed that JAK2 and STAT3 mRNA expression of control group and liposome group were higher than the SOCS3 group (P<0.05). This indicates that the excessive expression of SOCS3 can inhibit JAK2 and STAT3 produced. And more, the date showed SOCS2 mRNA expressive was suppressed by excessive expression of SOCS3.