The Construction Of SelS Gene Recombinant Plasmid And Its Protection Of Vascular Endothelial Cells From Hydrogen Peroxide-induced Injury

Objective A major death cause of type 2 diabetes mellitus (T2DM) patients is macrovascular complications. Atherosclerosis (AS)is the pathological base of macroangiopathy . Oxidative stress plays an important role in causing AS. Tanis is a newly detected protein and can affect carbohydrate metabolism. Tanis in human body is called SelS. It can interfere with clearance of oxygen free radicals and refrain apoptosis of B cell in islets of pancreas. This study is to construct the recombinant retrovirus vector PLNCX2-SelS through cloning gene of sels ,obtain stable clone expression of SelS gene and investigate the suppressing effect of SelS to vascular endothelial cell(VEC) injury induced by hydrogen peroxide(H2O2),and can provide experimental base for the following research on SelS,function and search new target for preventing and curing T2DM and Macroangiophy. Methods SelS gene segment obtained from human fat tissue by RT-PCR, was sub-cloned into retrovirus vector PLNCX2 twice,then the obtained recombinant eukaryotic expressing plasmid PLNCX2 -SelS was identified with ClaI/XhoI digestion and sequence analysis. The target gene was transfected stably into VECs(ECV-304) with liposome. Through screening by G418 , Stable expression of human SelS in VECs was obtained ,and was verified by RT-PCR . Its expressing level was detected using β- actin as inter- reference. VECs were divided into three groups: non-transfected group ,empty plasmid group and transfected group. All the cells were cultured in fresh serum-free medium 1640 containing 0,100,200,300,400μmol/L H2O2 for 24 hours. Their proliferative activity was examined by MTT assay. Results Electrophoresis proved that total RNA extracted from fatty tissue is intact. DNA sequencing make it clear that cDNA sequence of SelS is the same as the sequence in Genebank. The XhoI/ ClaI digestion and sequencing results showed that SelS gene segment had been coloned into recombinant retrovirus PLNCX2. The plasmid PLNCX2-SelS was insert successfully into genome DNA of VECs by PCR. The ratios of OD between SelS and β-actin in each group were 0.8512±0.3169,0.4136±0.2486 and 0.4324±0.2960 respectively, SelS mRNA level in transfected group was higher than in non-transfected group and empty plasmid group(P<0.01).The results of MTT assay showed that H2O2 inhibited the proliferation of cell through a kind of H2O2 concentration-dependent manner. Compared with non- transfected group, empty vector does not interfere with the growth of VECs(P=0.631), while the transfected SelS gene can weaken the inhibition of H2O2 against VECs ( P <0.05),especially the concentration of H2O2 were 300 and 400μmol/L. Conclusions We succeeded in cloning SelS gene and constructing PLNCX2-SelS eukaryotic expressing vector and obtaining high-efficiency stable clone in VECs. H2O2 can inhibit the proliferation of VECs in a H2O2 concentration- dependent manner. SelS can protect VECs from H2O2- induced injury.