Rnai Silencing Of Cox-2 On The Hsc Cell Kinetics And Lipid Metabolism Influence

Part I The construction of COX-2short hairpin RNA expres-sion vector and its effect on the inhibition of hepatic fibrosis.ObjectiveTo construct the short hairpin RNA eukaryotic expression vector for COX-2gene and observe its effect on the inhibition of hepatic fibrosis.Methods1.One rat COX-2gene and two human COX-2genes were chosen from the TRCand origene Genebank database, and were used for constructing three pairs of COX-2shRNA eukaryotic expression vector. Then these expression vector were identified byDNA sequencing and restriction enzyme digestion;2.The recombinant plasmids were transfected into the cell line HSC-T6byliposome and its effect of expression on COX-2mRNA and protein were detected byRT-PCR and Western Blot respectively. And fluorescence microscope was used toobserve transfecting efficiency;3.The adenovirus mediation recombinant plasmid were transfected into the SDrats vin tail vein, Rats were killed at the end of the12th week for taking livers. Theexpression of COX-2mRNA and protein in livers were detected by RT-PCR andimmunohistochemistry method respectively. Extent of liver fibering was analyzed byHE,MASSON staining. Image analyzer measured the area of liver fibrosis.Results1.The restriction enzyme digestion, DNA sequencing and alignmentdemonstrated that the three successfully inserted sequences of COX-2shRNA wereexactly correct;2.The transfecting efficiency of the three COX-2shRNAs exceeded70%byfluorecence microscope; 3.RT-PCR and Western-Blot analysis confirmed that all transfected COX-2shRNAs could inhibit the expression of COX-2gene in different extent,and COX-2shRNA-1group was the most efficient in all the groups;4.After being transfected into SD rat COX-2shRNA-1could inhibitthe expression of COX-2gene and hyperplasia of fibrosis tissue by RT-PCR andimmunohistochemical method.Conclusions1.COX-2shRNA eukaryotic expression vectors were constructed successfully;2.COX-2shRNA1,2,3have inhibiting effect on expression of COX-2inHSC-T6cells, and the effect of COX-2shRNA-1is the strongest;3.COX-2shRNA1could inhibit the expression of COX-2in SD rat liver tissue;4.COX-2shRNA1could down regulate the liver fibering in SD rat. Part II The influence of RNAi silencing COX-2on the geneexpression profiles of HSCObjectiveTo investigate the change of gene expression profiles after COX-2small hairpinRNA-1silencing COX-2in hepatic stellate cells.Methods1.The experiment is divided into COX-2shRNA1groups and HK groups;2.The extraction of total RNAs in HSC carried out by Trizol.And all the RNAswere purified into mRNA and then convened them to double-stranded cDNAs byreverse transcription;3.The fluorescence labeled cDNA was hybridized to27K Rat Genome Array.After strict film processing and by GenePix40OOB scanner scanning the arrayfluorescent signal images, software dealing with data, comparing the results,differential information of gene expression between them were acquired. Results1.After48hours and72hours of silencing COX-2in hepatic stellate cells, therewere45and93genes expressing in difference respectively (Ratio≥1.5and≤0.667);2.After48hours and72hours of silencing COX-2in hepatic stellate cells, therewere12and23genes expressing in difference respectively (Ratio≥2.0and≤0.5);3.The differentially expressed genes silenced by COX-2small hairpin RNA aremainly related to biological processes (54.32%,44.29%), molecular function(32.10%,37.14%) and cellular component at48hours and72hours respectively;Conclusions1.After hours of silencing COX-2, gene expression profiles of HSC changed;2.After hours of silencing COX-2, the differentially expressed genes are mainlyrelated to biological processes, molecular function and cellular component. Part III The influence of RNAi silencing COX-2on the cytody-namics and the expression of related genes in hepatic stellatecellsObjectiveTo investigate the influence of COX-2silencing small hairpin RNA(shRNA) onthe proliferation, apoptosis, differentiation,cell cycle of HSC, and demonstrate resultfrom gene arrays is in line with differential expression gene, which is closely relatedto cytodynamics.Methods1.Cell growth was measured by MTT assay in vitro;2.Apoptosis and cell cycle was examined by flow cytometry;3.The different expressed genes, closely related to cytodynamics, were detectedby RT-PCR and Western blot analysis.Results1.The apoptosis rate was significantly higher (89.27%±1.63vs23.85%±1.20vs10.23±1.34, both P <0.01) in the COX2-shRNA group than that in the HK(negative)and NC(blank control)groups at48h; 2.Proliferation of HSC-T6cell was inhibited remarkably at72h (P<0.05);3.G1%in COX-2shRNA1group is higher than that in HK(negative)and NC(blank control)groups;4.RT-PCR test shows Cdc27, Sh3kbp1expression was increased, and Plcd4wasdecreased. The former was in agreement with the chip;the latter was not. The restwere not significant.Conclusions1.COX-2shRNA1can downregulate proliferation of HSC-T6cell in vitro;2.COX-2shRNA1can promote HSC-T6cell apoptosis;3.COX-2shRNA1may delay cell cycle in G0/G1phase;4.RT-PCR test shows Cdc27, Sh3kbp1expression was increased, and Plcd4wasdecreased. Part IV The effects of RNAi silencing COX-2on lipometaboli-sm and the expression of related genes in hepatic stellate cellsObjectiveTo investigate the effects of COX-2small hairpin RNA(shRNA) silencing COX-2on lipid metabolism and demonstrate result from gene arrays is in line with ifferentialexpression gene, Which is closely related to lipid metabolism in hepatic stellate cells.Methods1.The content of triglyceride, cholesterol, vitamin A are detected by highperformance liquid chromatography (HPLC) at24h,48h and72h;2.The different expressed genes of lipid metabolism are detected by27K RatGenome Array48h and72h in cells and are confirmed by quantitative RT-PCR. Results1.The content of intracellular triglycerides increased by time(F=34.524,P=0.000)in COX-2ShRNA1group,and it is significantly different in COX-2ShRNA1group in contrast with that in the HK(negative)and NC(blank control)groups eachtime(P<0.05);2.The content of intracellular cholesterol decreased by time(F=360.808,P=0.000)in COX-2ShRNA1group,and it is significantly different in COX-2ShRNA1group in contrast with that in the HK(negative)and NC(blank control)groups eachtime(P<0.05);3.The content of intracellular vitamin A increased by time(F=19346.057,P=0.000)in COX-2ShRNA1group,and it is significantly different in COX-2ShRNA1group in contrast with that in the HK(negative)and NC(blank control)groups eachtime(P<0.05);4.RT-PCR confirmed the expression of Acsl6, Apol9a enhanced.Conclusions1.After RNAi silencing COX-2, content of triglycerides and vitamin A increasedand content of cholesterol decreased in cells;2.After RNAi silencing COX-2, expression of Acsl6, Apol9a enhanced.