The Effects And Mechanism Of Sulfatase-1Gene And Its N-terminal Active Fragment In Inhibiting The Activation Of Hepatic Stellate Cells

【Background＆Objective】The health and life quality of Chinese are in a great threat caused by liver fibrosis, as weare living in a big nation with the most people with hepatitis B around the world. More andmore alcohol abuse-related liver cirrhosis comes out, and there also exists numerous livercirrhosis patients in schistosoma japonicum suffering zone in China. The pathologicalprogressive process of liver fibrosis is characteristic with the course from liver fibrosis to livercirrhosis, and finally to liver cancer, implying us that we can stop the happening of livercancer by blocking the developing process of liver cirrhosis, and destroy the cancer nestsbefore its formulation. While it is still a worldwide problem to treat liver cirrhosis, there hasnot been an effective way to block the pathological process of liver fibrosis. More and moreevidence has shown the key factor to promote the liver fibrogenesis is the contribution of theinflammatory factors and cytokines involved in the micro-environment in liver fibrogenesis,and the most important factors include these growth factors: TGF-β1, PDGF, CTGF, FGF andVEGF. All of these growth factors need a combination with the specific receptors on cellsurface as well as the existence of heparin sulfate on HSPG to provide co-receptors andspatial configuration to stimulate the down-stream signaling in the cell. According to thenewly data, the sulfatase encoded by the gene Sulf-1is capable of desulfating the heparinsulfate on HSPG, then with the potential to stop the cell signaling of growth factors. Thus, wehypothesis that the Sulf-1is with the capacity to stop the pathological process of liverfibrogenesis by block the cell signaling of growth factors. The key role of the cells involved inliver fibrogenesis is hepatic stellate cells. So, inspired by the hypothesis, we detected thedynamic expression of Sulf-1gene in the activation of hepatic stellate cells, made an effort tothe construction of adenovirus vector of hSulf-1and plasmid vector of NSulf1, and theshRNA of hSulf-1, for the overexpressing and silencing of Sulf-1gene, in order to test theindex of liver fibrogenesis in LX-2and HSC-T6cell lines when Sulf-1is overexpressing orsilencing. We made a convincing proof to demonstrate that Sulf-1gene could ameliorate theactivation of hepatic stellate cells and the secretion of collagens. Simultaneously, for theexploring of an effective virus vector for gene therapy, enhancing the penetrating ability of thevector by reducing the molecule weight, we also constructed a plasmid vector of theN-terminal active fragment of hSulf-1gene, demonstrating the same effect ofanti-liver-fibrosis as the full length gene vector, laying a solid foundation for the exploring ofgene therapy regimen for anti-liver-fibrogenesis. 【Method】1、 Isolation of primary hepatic stellate cells and detecting the dynamic expression of Sulf-1gene in the activation of hepatic stellate cells.(1) Perfusion in situ and density-gradient centrifugation are adopted to isolate the primaryhepatic stellate cells in Wistar rats. Make a count for the yield of primary cells in each rat,test the vitality of the cells by TrypanBlue method.(2) Detect the expressing of α-SMA、Desmin、GFAP、F4/80in the isolated cells bywestern-blot, for the purpose of identifying of primary hepatic stellate cells.(3) Check the expressing of Sulf-1gene by RT-PCR on the culturing days0,3,5,72、 Test the mitogenesis-ptomoting effects of recombinant TGF-β1factor on LX-2andHSC-T6cell lines, in order to discover the best time-period and concentration ofrecombinant TGF-β1factor for stimulation.(1) Test the mitogenesis-ptomoting effects of recombinant TGF-β1factor on LX-2andHSC-T6in different time-period.(2) Test the mitogenesis-ptomoting effects of recombinant TGF-β1factor on LX-2andHSC-T6in different concentration.3、 Make a construction of adenovirus vector of Sulf-1and plasmid vector of NSulf1, and theshRNA of Sulf-1, for the overexpressing and silencing of Sulf-1gene. Using the affectionand transfection to establish the overexpressing and silencing of Sulf-1gene state in LX-2cell line.(1) Affect the LX-2cell line with different MOI of adenovirus vector Ad5-hSulf1and checkthe expressing of Sulf-1gene by PCR on different MOIs.(2) Transfect the LX-2cell line with hSulf1-shRNA, confirming the silencing of hSulf-1gene by RT-PCR.4、 Evaluate the index of liver fibrogenesis in LX-2and HSC-T6stimulated by recombinantTGF-β1factor when hSulf-1gene is overexpressed or silenced, validating theanti-liver-fibrogenesis effects of NSulf-1.(1) Using the affection of Ad5-hSulf1and transfection of hSulf1-shRNA to establish theoverexpressing and silencing of Sulf-1gene state in LX-2and HSC-T6cell linesstimulated by recombinant TGF-β1factor, check the expressing of α-SMA, CollagenⅠ,CollagenⅢ and TIMP-1by PCR.(2) Using the affection of Ad5-hSulf1and transfection of pDC315-NSulf1to establish theoverexpressing state of Sulf-1gene in LX-2and HSC-T6cell lines stimulated by recombinant TGF-β1factor, check the expressing of α-SMA, CollagenⅠ, CollagenⅢand TIMP-1by RT-PCR, check the expressing of α-SMA, CollagenⅠ, CollagenⅢ,p-ERK、p-AKT、p-Smad2by Western-blot.【Results】1、 As the Western-blot results shows, we have isolated the primary hepatic stellate cellssuccessfully, the yields of primary hepatic stellate cells in each rat is (3.008±0.3)×107perrat, and the vitality of the primary hepatic stellate cells is (95.80±1.9)%on average. TheRT-PCR results showed that the expression of hSulf-1gene is gradually down-regulatedin the activation process of primary hepatic stellate cells.2、 The best condition to stimulate LX-2with recombinant TGF-β1factor is:5ng/ml，24hours and the best condition to stimulate HSC-T6with recombinant TGF-β1factor is:7.5ng/ml，24hours.3、 The best MOI to affect LX-2with Ad5-hSulf1is50, and the expression of hSulf-1genecould be silenced with transfection of hSulf1-shRNA in LX-2.4、 As the PCR and Western-blot results showed, hSulf-1and NSulf-1could ameliorate theexpression of α-SMA, TGF-β1, CollagenⅠ, CollagenⅢ, TIMP-1, p-ERK, p-AKT,p-Smad2. Silencing of hSulf-1in LX-2does not show an obvious impact on theexpression of the indexes of liver fibrosis.【Conclusion】In our study, we successfully isolated the primary hepatic stellate cells, proved thedynamic decreasing expressing of Sulf-1gene in the activation process of culturing primaryhepatic stellate cells. We successfully constructed the adenovirus vector for hSulf-1gene andthe plasmid vector of its N-terminal active fragment NSulf-1, as well as the shRNA of hSulf-1gene for the overexpressing and silencing of hSulf-1gene. We investigated theanti-liver-fibrosis effects of hSulf-1gene in LX-2and HSC-T6cell lines stimulated byrecombinant TGF-β1factor, made a strong evidence to elucidate that hSulf-1gene and itsN-terminal active fragment NSulf-1could ameliorate the activation of hepatic stellate cellseffectively as well.