| Objective:To observe the influence of SHBs protein to liver cells andsome genes’ expression such as ECHS1,PPARa and its downstream genes,provide n ew clews to see SHBs’ biological function,and screen the fatty liver formation mechanism in HBV infected persons.Methods:Polymerase chain reaction(PCR) was applied to amplify the SHBs gene used C-type HBV full-length S gene as template.The target gene SHBs was cloned into pcDNA3.1(+) vector.First defined the best G418screening con centration in HepG2cells,then transfected the recombinant plasmid pcDNA3.1-S HBs and empty plasmid into HepG2cells respectively,afterwards acquired the t wo stably transfected cell lines HepG2-pn3.1-SHBs and HepG2-pn3.1-neo throu gh G418screening.The SHBs mRNA and protein in the stably transfected cell lines were detected by RT-PCR and ELISA.Real-time quantitative PCR and We stern-Blot were used to detect some genes’mRNA and protein expression differe nces in connection with lipid metabolism between those two cell lines.Results:The eukaryotic expression plasmid pcDNA3.1-SHBs was constructe d successful.The best G418screening concentration in HepG2cells is1000ug/ml. The stable transfection cell lines HepG2-pn3.1-SHBs which continually express ing target SHBs protein and HepG2-pn3.1-neo containsempty plasmid were esta Wished successfully.The target protein SHBs in HepG2-pn3.1-SHBs cell lines w asvalidated correctly by RT-PCR and ELISA.The stably transfected cell lines H epG2-pn3.1-SHBs express SHBs protein continually.Compared with the HepG2-pn3.1-neo cells,HepG2-pn3.1-SHBs cell has much higher content of the two tra nsaminases ALT and AST content in culture supernatants[(46.80±4.78)vs (39.21 ±2.81);(64.72±5.45) vs(54.58±1.20)(p<0.01)],much higher CHO content in Ce11Lysis Solution’[(0.87±0.04)vs (0.87±0.04),p<0.01)],and lower TG levels [1.39±0.65) vs (0.58±0.05),p<0.01)].Real-time quantitative PCR results showed that: Compared with the HepG2-pn3.1-neocells, the transcription levels of the gene ECHS1,APOAland LPL in HepG2-pn3.1-SHBs cells were significantly reduced [(0.161±0.043)vs(0.210±0.022),(p<0.05);(0.031±0.007)vs(0.094±0.055),(p<0.05);(0.770±0.036)vs(0.982±0.031),(p<0.001)];CPT1a and PPARa genes’ transcrip tional level differences weren’t statistically significant,but showed signs of down grading[(0.028±0.005)vs(0.030±0.004);(0.014±0.004)vs(0.015±0.002)];ACC and S REBP-lc’s transcriptionwere enhanced significantly[(0.113±0.027)vs(0.059±0.022),(p<0.01);(0.019±0.002)vs(0.015±0.001),(p<0.01)].Western-Blot results showed tha t the trend of these genes’ protein levels in accord with mRNA.Conclusion:The results show that the SHBs can cause some damages to liver cells and interfere fatty acid metabolism,and affect some genes’ expression in connection with fatty acid synthesis and decomposition.But it is not clear whether HBsAg directly result in hepatic steatosis, it is worthy of further stud y. |
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