The Effect And Mechanism On Selenoprotein S Protecting Endothelial Cells

Endothelial cell dysfunction(ECD) plays an important role in development of diabetic vascular complications.More and more researchers are interested in how to pretect endothelial cell effectively.The purposes of this study are shown as the followings:(1) To study on correlation between SelS mRNA expression in Chinese adipose tissue,Insulin resistance(IR) and Serum Amyloid A(SAA),which provides a new direction for preventing the development of type 2 diabetes mellitus(T2DM) and atherosclerosis.(2) To construct the Eukaryotic expression vector of Selenoprotein S(SelS) and transfect into human umbilical vein endothelium cells (HUVECs,ECV₃₀₄ Cells) by using molecular biological technology and identify the DNA sequence analysis,mRNA and protein level of SelS,which provides a reliable technique support for SelS study.(3) To establish the cellular damage model induce by exogenous hydrogen peroxide(H₂O₂) and study the over-expression of SelS in the protective function and mechanism of ECV₃₀₄ cell,which provides a new strategy for preventing the development of T2DM and atherosclerosis.The following studies of human omental adipose tissues from ten cases of type 2 diabetic patients and twelve cases of non-diabetic individuals were carried on to examine the expression level of Tanis/SelS mRNA by semi-quantitative PCR, calculate Homa-IR by standard formula and detect SAA level by ELISA.Tanis/SelS mRNA expression,Homa-IR and serum SAA were higher in T2DM than in control group.Tanis/SelS mRNA level was positively correlated with Homa-IR and SAA level in two groups.SelS protein might involve in insulin resistance in Chinese with T2DM,possibly acting as the SAA receptor,thus play an important role in the development of T2DM and atherosclerosis.After total RNA was isolated from human subcutaneous adipose tissue,SelS gene segment,which was called SelS Insert DNA1,was amplified by RT-PCR and then cloned into the vector of pMD18-T.Recombinant plasmid of pMD18-SelS was confirmed by DNA sequence analysis.SelS Insert DNA2,which was purified successively by plasmid amplified,restrictive digestion and gel extraction,was subcloned into Eukaryotic expression vector of pLNCX2.After identified by PCR and DNA sequence analysis,the recombinant plasmid of pLNCX2-SelS was transfected into ECV₃₀₄ cell and SelS were expressed successfully both in mRNA and protein levels.Moreover,the gene expression level of SelS was 1.76 fold in transfection group compared to endogenous gene expression and the protein expression level was 1.56 fold.After ECV₃₀₄ cells were damaged by different concentration of H₂O₂,both of the activity of cellular proliferation and SOD were increased in the group of over-expressed SelS gene compared with control one.Furthermore,elevated SelS gene expression in ECV₃₀₄ cells could attenuate the production of MDA and inhibit the up-regulation of Caveolin-1 induced by H₂O₂.It is suggested that SelS might protect endothelial cells from H₂O₂-induced impairment through anti-oxidative pathway and this protective mechanism might related to the depressing the elevation of Caveolin-1 by SelS.Therefore,up-regulated SelS in adipose might involve in insulin resistance, possibly acting as the SAA receptor,thus play an important role in the development of T2DM and atherosclerosis.SelS gene fragment isolated from human subcutaneous adipose was inserted into retrovirus vector of pLNCX2 and recombinant Eukaryotic expression vector of pLNCX2-SelS was successfully constructed.Elevated SelS, which was also expressed endogenously in ECV₃₀₄ cells,could attenuate the damage induced by H₂O₂ and play a role in ECV₃₀₄ cellular anti-oxidative protection through preventing the elevation of Caveolin-1.