Effect Of RNA Interference Targeting Leptin Gene On Biological Character Of Activated Hepatic Stellate Cells

Part oneThe expression of leptin in hepatic fibrosisObjective:To study the clinical and significance of leptin in pathogenesis of hepatic fibrosis.Methods :The immunohistochemical ABC method and hematoxylin-eosin were used to determine the leptin expression in 10 cases of normal liver and 20 of hepatic fibrosis. Result :The results revealed that the leptin existed in livers . The expression of leptin in hepatic fibrosis was significantly higher than that of in normal liver (P<0.01).epithelial cell of bile capillary and hepatocyte and lipocyte were leptin positive.The density of positive cells in hepatocyte were widly expressed in epithelial cell of bile capillary. Conclusion :There is relationship between the expression of leptin in liver and the pathogenesis of hepatic fibrosis . These results might indicate that leptin participated pathology in liver fibrosis .Part twoThe construction of screening platform for RNA interferencetargeting leptin geneObjective: To construct the specific siRNA expression vector and screen the effective recombinant leptin siRNA plasmid vectors in eukaryocyte.Methods : The seven siRNAs against leptin gene were synthesized intracelluarly by expressed templates of plasmid vector psiRNA- hH1neo. The recombinant leptin siRNAs plasmid vectors could be expressed in eukaryocyte, and then to evaluated them by using enzyme cutting and sequencing. The recombinant plasmids were transfectedinto HSCs using this construct via Lipofectamine methods separately. The cell adhesionwas determined with MTT assay.Result: Identification by enzyme cutting and sequencing showed that the siRNAexpression vectors targeting leptin were constructed successfeully; The cell adhesiondeclined markedly compared with no-transfected HSC. Among siRNAs, siRNA278,siRNA108, siRNA97 and siRNA454 had stronger inhibition.Conclusion : The recombinant leptin siRNA278, siRNA108,siRNA97 and siRNA454could attenuate cell adhesion.Part threeEffect of RNA interference targeting leptin gene on biologicalcharacter of activated hepatic stellate cellsObjective: To study the expression of siRNA vector against leptin and the effect onbiological character of activated HSCs.Methods : HSC cells were transfected with the eukaryotic constructs encodingleptin,then the stable cell clones were selected for the analysis of their performances.The effect of siRNAs were evaluated through the analysis of proliferation, activation,collagen deposition of HSC.Result : siRNAs down-regulated leptin expression in activated HSCs. The furtherstudies demonstrated that siRNAs reduced deposition of collgen I, suppressed HSCproliferation and HSC activation in activated HSCs.Conclusion : The siRNAs against leptin designed in this study reversed the character ofactivcted HSCs by down-regulating leptin expression efficiently and diminishing leptinsignaling underlying HSC activation. As a consequence, it would provide as a novelcandidate of therapeutic agents for hepatic fibrosis in further study. Part fourThe effect of siRNAs targeting leptin on signal transduction in hepatic fibrosisObjective : To explore the signal transduction induced by siRNAs targeting leptin inhepatic fibrosis.Methods: The siRNAs were transfected into HSCs using Lipofectamine methodsrespectively. The cells were selected after growing in DMEM containing 300μg/mlG418 for about 4 weeks. Gene expression of p-STAT3 and TGFpi were showed byWestern Blot analysis and reverse transcription polymerase chain reaction(RT-PCR).The immunohistochemical ABC method was also used in experiment.Results: The expression of p-STAT3 and TGFpi were decreased after siRNAstransfection into cultured HSC compared with control group.Conclusions: p-STAT3 and TGFpi maybe involved in leptin signal transductionpathway in HSC; the effect of siRNA on transduction of HSC provide a new theorymethod for hepatic fibrosis.Part five Inhibition of α-interferon and siRNAon proliferation of hepatic stellate cellObjective: Recently, the research showed that siRNA could inhibit hepatic stellate cell. The study was designed to investigate the effects of a-interferon and siRNA97 on activity of hepatic stellate cells.Methods: The recombinant plasmid targeting leptin gene coding sequence(psiRNA97) was constructed successfully. HSCs were treated with a-interferon and siRNA97.The growth curve, soft-agar colony-forming rate were used to analyze the inhibition to cell proliferation.Collagen type I ,III contents in cell media were detected by ELISA.The cell cycle was detected by flow cytometry(FCM). Telomerase activity was determined by PCR-EL1SA methods.Results: α-interferon and siRNA97 markedly inhibited proliferation of HSC (P<0.05) , and reduced the colony-forming rate in the soft-agar drastically (P<0.05) ; FCM showed that the cell cycle was arrested in the G1/G0 phase; compared with control group, collagen type I ,III contents in cell media were significantly decreased (P<0.01). Telomerase activity was dramatically declined during the cell incubation with a-interferon and siRNA97.Conclusion: Combination of a-interferon and siRNA97 has anti-fibrosis activity in vito compared with HSC treated with a-interferon and siRNA97.Part sixEffect of octreotide in the regulation of intracellular free Ca²⁺concentration and cell proliferation of hepatic stellate cellObjective:To investigate the effect of octreotide on the regulation of intracellular free([Ca²⁺]_i and cell proliferation of hepatic stellate cells(HSCs).Methods: To observe the content of Ca²⁺ in HSCs by the fluorescence Ca²⁺ indicator Fura-2/AM in normal and chronic hypoxic condition. The influences of octreotide on HSCs proliferation were assessed by MTT assay and the level of cAMP and cGMP with radioimmunology assay.Results: The content of [Ca²⁺]j in hypoxic condition was markedly increased as compared with normoxic condition (P <0.01) . It was (137.7±7.8 ) and (293.2±12.4) nmol/L, respectively. In normoxic conditions, the content of [Ca²⁺]j decreased sharply after 500, 800 and 1000μg/L octreotide treatment, it was (92.52±2.52), (83.77±2.30 ) and (76.58±2.21) nmol/L, respectively, (P <0.01). In hypoxic condition, octreotide 500, 800 and 1000μg/L caused the reducation in [Ca²⁺]_i. it was (204.28±7.41) (174.08±4.77) and (156.75±6.59)nmol/L, respectively, (P <0.01). MTT assay showed that octreotide 500, 800 and 1000μg/L reduced the value of absorbing light degree (A value) in normoxic and hypoxic condition. After exposure to hypoxia condition, the level of cAMP was not significantly different from cGMP (P >0.05). The contents of cAMP and cGMP increased (P <0.05) after octreotide 500μg/L treatment in normoxic and hypoxic condition. Moreover , the contents of cAMP and cGMP increased greatly (P <0.01) after octreotide 800,1000μg/L treatment in same condition.Conclusion:Hypoxia can promote the proliferation of HSC through the second messenger system and octreotide antagonizes the action dependent dosage in hypoxic and normoxic conditions .cAMP and cGMP might regulate HSC in hepatic fibrosis.