The Impact Of HBV Pre S2 Protein On The Signal Molecule Lysophospholipids Acid And The Effect Of LPA On Insulin Secretion

Objective To investigate the expression and the nucleus localization of hepatitis B virus(HBV) pre-S2 protein(pre-S2) and the trans-activation of pre-S2 on the promoter of lysophospholipase A-1(LYPLA-1), which can hydrolyze lysophosphatidic acid(LPA), examine the effect of exogenous LPA on insulin secretion of islet cells, and thus provide research clue and experimental basis for further study of the mechanism underlying the diabetes complication of hepatitis B. Methods 1. The expression and the nucleus localization of HBV pre-S2(1)Construction of the expression vector of HBV pre-S2 and green fluorescent protein(GFP) fusion gene: HBV DNA was extracted from the sera of hepattis B patients, the gene of HBV pre-S2 was amplified using the specific primers with Hind Ⅲ and Eco RⅠcutting sites and inserted, after recovering it from the gel, into the T-easy vector to construct pGEM-HBV-S2. The pGEM-HBV-S2 and pcDNA3.1-EGFP were digested with Hind III and EcoR I, and the fragments of the pre-S2 and the digested pcDNA3.1(+)-EGFP plasmid were gel-recovered, respectively. The expression vector of HBV pre-S2 and GFP fusion gene, pcDNA3.1-PreS2-EGFP, was obtained by ligating the two fragments and transforming Ecoli DH5α with the ligated product.(2) pre-S2 expression in nuclei: HepG2 cell was transfected with pcDNA3.1-PreS2-EGFP and the nuclei was stained with 4’,6-diamidino-2-phenylindole(DAPI) after 48 h of transfection. The pre-S2 expression in nuclei was determined by observing the location of GFP using a fluorescence microscope.2. The trans-activated effect of pre-S2 on the promoter of LYPLA-1 HepG2 cells were co-transfected with the luciferase reporter plasmid of LYPLA-1 promoter and the eukaryotic expression plasmid of HBV pre-S2, and the transcriptional activation activity of pre-S2 on LYPLA-1 promoter was evaluated by examining the relative activity of luciferase.3. The effect of exogenous LPA on insulin secretion.(1)The isolation and purification of the islet cells of mice: the mouse pancreatic islet was digested by retroperfusion of collagenase V via common bile duct, followed islet isolation, and then islet cells were purified with the centrifugation of discontinuous density gradient of Ficoll.(2) The effect of exogenous LPA on the insulin secretion of the islet cells: the primery islet cells and the insulin-secreting cell MIN6 were stimulated with 0μmol/L, 1μmol/L, 2μmol/L, 3μmol/L, 4μmol/L and 5μmol/L of LPA and the insulin secretion was determined by assaying the insulin concentration calibrated with total protein concentration. Results DNA sequencing results indicated that the recombinant vector pcDNA3.1-PreS2-EGFPcontains the sequence of the pre-S2 gene, and enzyme digestion and sequencing after subcloning confirmed that the constructed recombinant vector is consistent with the design. The result of observation with the fluorescence microscope displayed that pre-S2-EGFP fussion protein expressed and the green fluorescent protein was located in nuclei after pEGFP-preS2 transfection. HBV pre-S2 activated LYPLA-1 promoter. The insulin secretion was affected by exogenous LPA, and the maximum amount of the insulin secretion was at 3μmol/L of LPA. Exogenous LPA enhances the insulin secretion of insulin-secreting cells in the range of 0-3μmol/L of LPA. Conclusions HBV pre-S2 can locate in nuclei and activate the promoter of LYPLA-1 which can hydrolyze LPA, and the exogenous LPA impacts the insulin secretion secretion of islet and MIN6 cells and enhances the insulin secretion of the cells in the range of 0-3μmol/L of LPA.