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Effects of Expression of SUMO-1in Rat Hepatic Stellate Cells Which was Transfected by Overexpression and Si-RNA Lentiviral Vector

Author: WangFuChang
Tutor: GuoWuHua
School:
Course: Oncology
Keywords: liver fibrosis lentiviral HSC-T6 SUMOs cell cycle and apoptosis
CLC: R575.2
Type: Master's thesis
Year: 2013
Downloads: 24
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Abstract


Hepatic fibrosis(HF) is a common clinical and pathological syndrome due tochronic liver diseases. The expression of many genes is anomalous during the processof hepatic fibrosis, and there is increased risk to develop to hepatocellular carcinoma.Activation and proliferation of hepatic stellate cells (HSCs) is the core of hepaticfibrosis. Hepatic stellate cellsted which are located in the perisinusoidal Di.sseclearance, under normal state, main storage and metabolism of vitamin A, synthesisand secrete a small amount of extracellular matrix (extracellular matrixc, ECM).Hepatic stellate cellsted which are located in the perisinusoidal Di.sse clearance,under normal state, main storage and metabolism of vitamin A, synthesis and secretea small amount of extracellular matrix (extracellular matrixc, ECM). When HSCswere stimulated and activated excessively, it can change into be myofibroblasts whichwas with proliferative activity and with the characteristics of shrinkage.Then it canexpress α-smooth muscle actin (Alpha smooth muscle actin, α-SMA). So it is thatabnormal increase of ECM which escesses hepatic clearance capacity results inintrahepatic accumulation is the important mechanism of formation of liver fibrosis.Studies have shown that proper concentration of LPS may stimulate HSC to enhancethe proliferation rate and the expression of intracellular collagen enhances. Inductionof LPS cell model can successfully simulate the liver fibrosis formation process ofHSC in the number and functional aspects of the major changes.So HSC activationstudies have provided a more comprehensive cell model. Our research with liverfibrosis degree deepening, the increased expression of SUMO-1.small ubiquitin-related modifier (SUMO) has four gene family members,whichcan regulate intracellular target proteins distribution and its function through thecovalent modification of target proteins,and protein interactions.With liver fibrosisdegree deepening, the increased expression of SUMO-1has been found. SUMO-1may make TGF-β1and PDGF expression enhance, which can promote theproliferation and activation of HSC, promote the secretion of large collagen, andreduce collagen degradation leading to a great deal of abnormal deposition of collagen. SUMO-1can modify other growth factors, to maintain the stability ofprotein, and regulation of liver fibrosis and liver development process. Therefore, wedesign the experiment about mechanism of SUMOs during the process of liverfibrosis. This experiment first detected the expression level changes of SUMO-1ofHSC-T6which was stimulated LPS by Western blot and quantitative PCR in orderto further understand the funtion of SUMO-1in the progress of hepatic fibrosis. Afterthe HSC-T6which was activated by LPS was transfected by the lentivirus aboutSUMO-1and was confirmed that the expression level of SUMO-1did getinterference and up-regulation by Western blot and quantitative PCR, we detected thecell cycle and apoptosis the HSC-T6cell line by flow cytometry in order to expoundthe mechanism of SUMO-1in hepatic fibrosis.Part I Expression level of SUMO-1of HSC-T6which wasstimulated by the LPS of different concentrationsObjectiveTo investigate the optimum stimulation concentration of LPS on HSC-T6in thesimulation of hepatic fibrosisMethodsThe final concentration of LPS was divide into0ug/ml,1ug/ml,10ug/ml,100ug/ml,200ug/ml. After the LPS of stimulation on HSC-T6for24hours,eachgroup of RNA and protein were extracted,which was usd for quantitative PCR andWestern blotting to detect the expression level of SUMO-1and for Western blottingto detect the expression level of α-SMA.ResultsThe result of quantitative PCR found that the group of the concentration of10ug/ml LPS expressed the highest levels of SUMO-1, and the difference wasstatistically significant (P<0.05). Western blotting verificated the rule andexpression ofα-SMA is consistent with SUMO-1, and the difference was statisticallysignificant (P<0.05).ConclusionLPS can effectively activate HSC-T6Within a certain range. At the concentration of10ug/ml LPS of stimulation on HSC-T6can best simulate ofhepatic fibrosis,which was content with the expression of SMUO-1. The resultindicates that SMUO-1may play a part in the activation process of HSC-T6.Part Ⅱ Study on the effect and mechanism of proliferation foHSC-T6after the expression of SUMO-1changesObjective:To discuss the role and mechanism of SUMOs during the activation ofHSC-T6Methods:After the SUMO-1siRNA lentiviral and SUMO-1-IRES2lentiviral weretransfected into HSC-T6, the level of SUMO-1was detected by Real time-PCR andWestern blot, the cycle and apoptosis of HSC-T6was tested by flow cytometry andthe express level of SUMO-2and SUMO-3was checked by Real time PCRResults:The siRNA could significantly silence the expression of SUMO-1in HSC-T6but the level of SUMO-2and SUMO-3was up-regulation.And the SUMO-1-IRES2promte the expression but the level of SUMO-2and SUMO-3wasdown-regulation.The differnce of the level of SUMO-1/-2/-3was statisticallysignificant which was compared with the control groups.The difference of the cycleand apoptosis of all groups was not statistically significant (P>0.05)Conclusion:The effect of SUMO-1lentiviral gene in HSC-T6is good, but the level ofSUMO-1was opposite wih SUMO-2and SUMO-3.And the cycle and apoptosis ofHSC-T6showed no obvious change. The results showed that each member ofSUMOs can be complementary with others in funtion and all together led to HSC-T6action

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Cirrhosis
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