Dissertation > Excellent graduate degree dissertation topics show

The Construction of SOCS3 Eukaryotic Expression Vector and Effection on Apoptosis of the Adipose Cells

Author: AnLei
Tutor: SunChao
School: Northwest University of Science and Technology
Course: Cell Biology
Keywords: SOCS3 Eukaryotic expression vector Apoptosis 3T3-L1 Preadipocytes
CLC: Q255
Type: Master's thesis
Year: 2011
Downloads: 33
Quote: 0
Read: Download Dissertation

Abstract

Cell signal transduction inhibitors (suppressors of cytokine signaling, SOCS) family of protein structure, composed of SH2 domain, N area and consisting of about 40 amino acids, the C-terminal SOCS box. Initially thought to be a negative feedback regulation factor to regulate the JAK / STAT signaling pathway, which SOCS3 Many studies have confirmed participation in leptin (Leptin), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), negative feedback regulation of growth hormone (GH) signaling pathway. In recent years, the in-depth study of SOCS3 that in cell growth, differentiation and apoptosis process also plays an important role in this study as the research object in 3T3-L1 cells and mouse precursor fat cells, which focused on SOCS3 adipocyte apoptosis. Prophase of mouse adipose tissue extracted RNA as a template, RT-PCR amplification of SOCS3 gene was cloned into pMD18-T, construct the recombinant plasmid pMD18-T-SOCS3 SOCS3 gene was amplified as a template to pMD18-T-SOCS3 pEGFP-N1-SOCS3 recombinant plasmid was cloned into pEGFP-N1, to construct eukaryotic recombinant plasmid pEGFP-N1-of SOCS3; the trial late to build successful transfection of 3T3-L1 cells and mouse precursor fat cells under a fluorescence microscope observed reporter gene GFP expression, RT-PCR was used to detect apoptosis related genes Bax, c-myc, bcl-2, of mcl-1, survivin, as well as the expression of JAK2 and STAT3, SOCS3 protein expression and Western blotting detection of intracellular preliminary study impact of SOCS3 the adipocyte apoptosis JAK2/STAT3 signaling pathway, the main findings are as follows: 1. cloned by PCR using mouse SOCS3 gene contains cds cDNA full-length sequence of length 678bp, Genbank No. No . NM ₀ 07707.3. SignalP 3.0 Server on the the SOCS3 amino acid sequence analysis, and were used to Neural Networks (NN), and Hidden Markov Models (HMM) analysis found the SOCS3 (cds) itself carries a signal peptide possibility is zero, is a non-secreted protein, and using SWISS-MODEL protein 3D structure prediction of SOCS3 cds. Mouse adipose tissue extracted RNA as a template, RT-PCR cDNA was cloned into pMD18-T and pEGFP-N1, to construct the recombinant plasmid pMD18-T-SOCS3 and pEGFP-N1-SOCS3 contains cds District of SOCS3 positive clones by restriction enzyme digestion and PCR, and sequencing proved SOCS3 properly inserted into the pMD18-T and pEGFP-N1 pMD18-T-SOCS3 and pEGFP-N1-SOCS3 by sequencing homology of 100%, was successfully constructed expression vector pEGFP- N1-SOCS3. 3. use to build correct pEGFP-N1-SOCS3 liposomes 3T3-L1 cells transfected with the control group and liposome group served as controls, found that liposome and SOCS3 fluorescence microscopy report gene GFP's expression can be observed; RT-PCR and by Western blotting were detected to the expression of cell SOCS3 mRNA and protein of significantly significantly improve (P lt; 0.05); by Hoechst 33258 staining found, of SOCS3 group of apoptosis significantly; of Bax and c-myc mRNA expression levels were significantly higher (P lt; 0.05), bcl-2 and of mcl-1 gene expression levels were significantly lower (P lt; 0.05). SOCS3 promote the occurrence of 3T3-L1 cells apoptosis. 4 build correct pEGFP-N1-SOCS3 liposomes transfection of mouse precursor fat cells as a control, with blank group and liposome group, found that liposome and SOCS3 under a fluorescence microscope observation to report gene GFP's expression; RT-PCR and by Western blotting detection to cells SOCS3 mRNA and protein expression significantly significantly improve (P lt; 0.05); of Bax, of c-myc and survivin gene's mRNA expression level significantly increased (P lt; 0.05), bcl-2, of mcl-1 and NF-kB gene expression levels significantly lower (P lt; 0.05). The results show that: SOCS3 promoted mouse preadipocyte apoptosis. 5 build correct pEGFP-N1-SOCS3 transfected by liposomes mouse preadipocytes with blank group and liposome group as a control detection JAK2/STAT3 signaling pathway gene expression, blank group and the liposome group JAK2 and STAT3 expression was significantly higher than that SOCS3 group (P lt; 0.05). This suggests that the overexpression of SOCS3 inhibited JAK2/STAT3 signaling pathway. In addition, the data also showed that overexpression of SOCS3 SOCS2 inhibited.

Related Dissertations

CLC: > Biological Sciences > Cell Biology > Cell physiology > Cell aging and death
© 2012 www.DissertationTopic.Net Mobile