Establishment Of An Immortalized Human Pancreatic Stellate Cell Line(HP-1) And Study On Its Biological Characteristics And Retinoic Acid Inhibiting Pancreatic Fibrosis

Activated pancreatic stellate cell(PSC)plays an important role in the pathogenesis of pancreatic fibrosis.In normal pancreas,PSCs are quiescent and can be identified by the presence of vitamin A lipid droplets in the cytoplasm.In response to stimulus signals,they are transformed from quiescent phenotype into myofibroblast-like cells,which lose vitamin A lipid droplets and express cytoskeleton proteins: alpha-smoth muscle actin(α-SMA),vimentin,desmin and glial fibrillary acidic protein(GFAP).Activated PSCs can produce fibrogenic cytokine transforming growth factor β1(TGF-β1)and mitogenic cytokine platelet-derived growth factor(PDGF),synthesize and secrete extracellular matrix(ECM)including type I collagen(Col1),and produce matrix metalloproteinases(MMP-1,MMP-2,etc)and tissue inhibitors of matrix metaloproteinases(TIMPs).TGF-β1 regulates PSC activation in autocrine and paracrine fashions,increases the expression of α-SMA and Col1,and down-regulates the synthesis of matrix metalloproteinase-9(MMP-9).Interleukin 6(IL-6)is an important inflammatory cytokine that promotes PSC activation.All-trans retinoic acid(ATRA)is an active vitamin A metabolite that induces rodent PSC into a resting state,reduces ECM synthesis,and inhibitsethanol-induced α-SMA expression.Therefore,PSCs have been implicated in the pathogenesis of pancreatic fibrosis and the reversal of ECM.Primary PSCs can’t be cultured for a long time due to the lack of pancreatic tissue and the presence of cell aging.The limited number of PSCs can’t meet its functional research.So far,a few human PSC lines have been established.These cell lines are difficult to be widely used because of its limited growth potential and shortage of the phenotypic characteristics of primary PSC.In this study,a stable immortalized human PSC line(designated as HP-1cell)was established by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated PSC.We assessed the possibility on studying PSC-mediated human pancreatic fibrogenesis by analyzing and comparing the biological characteristics and proteomics between HP-cell and PSC.Further more we established an ALC-treated HP-1 cell model to clarify the effect of ATRA on inhibiting ALC-induced PSC fibrosis and to elucidate the mechanism of ATRA on inhibiting TGF-β1 signaling pathway.Our study could be divided into two parts:Part One: Establishment of an Immortalized Human Pancreatic Stellate Cell Line(HP-1 cell)and Analysis of Its Biological Characteristics and ProteomicsObjective: The aim of this study was to establish an immortalized PSC line(HP-1 cell)by using RSV promoter/enhancer to drive the expression ofSV40 T antigen.We determine the level of matrix proteins and matrix remodeling regulatory proteins in response to stimulators and analyze the protein expression profiles of HP-1 cells versus PSCs to evaluate the possibility of HP-1 cells as a useful tool in the study of PSC-mediated pancreatic fibrogenesis.Methods: PSCs were isolated from normal pancreatic tissue resection samples around benign pancreatic occupying lesion by collagenase P perfusion digestion,and cultured in 10% FBS DMEM.The fourth generation of activated PSC was used in this study.A stable immortalized human PSC line HP-1 cell was established by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated PSC.The expression of SV40 T antigen and GFAP in HP-1 cells was detected by immunocytochemistry while the expression of vimentin,desmin and α-SMA was determined by immunofluorescence.The levels of α-SMA,CTGF,Col1A1,MMP-1,MMP-2 and TIMP-2 m RNAs in HP-1 cells and PSCs were determined by RT-q PCR.The protein contents of matrix proteins,matrix remodeling regulatory proteins and cell receptors including TGFβR2,Bambi,PDGFRβ and PPRPγ were detected by Western Blot.The green fluorescent protein plasmid p EGFP-N1 was transfected into HP-1 cells by lipofectamine Fu GENE 6 and X-Treme GENE Si RNA reagent,respectively.HP-1 and PSC cell lysis proteins were separated,digested by trypsinase,labeled by tandem mass tag(TMT),separated by EASY-n LC 1000,and submitted to high-efficiency MS/MS for detection and analysis.In thisXII study,1.5 fold change was used as the threshold of differential protein expression.T-test was used to determine the significant difference between the two kinds of cells.A value of P< 0.05 was considered statistically significant.Results: In this study,a stable immortalized PSC line HP-1 cell was established,which expressed SV40 T antigen in the nucleus and characteristic cytoskeleton protein(GFAP,vimentin,desmin and α-SMA)as well as cell recptors(TGFβR2,Bambi,PDGFR and PPRPγ)in the cytoplasm.Further studies showed that HP-1 cells were consistent with PSCs in increased expression of α-SMA、CTGF、Col1A1 and TIMP-2 m RNAs and proteins,and dicreased expression of MMP-1/2 m RNAs and proteins in response to TGF-β1.The transfection efficiency of the lipofectamine Fu GNE 6 and x-tremegene Si RNA for HP-1 cells was 61.8% and 88.65% respectively.Comparative proteomic study showed that there were 4,537 quantitative shared proteins between HP-1 cell and PSC.Any unique protein in each cell type alone wasn’t found.Statistical analysis showed 54 up-regulated proteins in HP-1 cells and45 up-regulated proteins in PSC(P<0.05)and the remaining 4438 proteins(97.8% of the total quantifiable proteins in the two cells)without significantly different.If the differential proteins are defined as > I±2.0-fold change I,they don’t contain extracellular matrix protein,fibrogenic cytokine,cell growth factor and matrix remodeling regulatory proteins.Conclusion: HP-1 cell has the characteristic markers of activated PSC.HP-1 cell is consistent with PSC in response to fibrogenic stimulator and inprotein expression profile,which can be used as an effective tool for the study of PSC-mediated pancreatic fibrosis.Part Two: Experimental Study of All-trans Retinoic Acid in Inhibiting PSC-Mediated Pancreatic FibrosisObjective: The aim of this study was to clarify the characteristics of ATRA in inhibiting human PSC-mediated fibrosis induced by ALC,and to evaluate the role of ATRA in anti-pancreatic fibrosis via inhibiting TGF-β1signaling pathway.Methods: A stable immortalized human PSC line HP-1 cell was established by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated PSC.In this study,a low-serum culture model was established from HP-1 cells.The formation of lipid droplets in HP-1 cells was detected by Bodipy fluorescence staining while the production of Smad2/p Smad2 and p Smad3/Smad3 was analyzed by Western blot.The levels of Col1A1,α-SMA,IL-6,TGF-β1 and MMP-9 m RNAs were determined by RT-q PCR.The protein contents of Col1A1,α-SMA,IL-6,TGF-β1 and MMP-9were detected by ELISA.Results: In this study,ATRA(5 μM)could significantly promote the formation of lipid droplets in the HP-1 cells that treated by low-serum(0.5%FBS)culture alone or by low-serum plus ALC(50 m M),thereby inducing the cell transition to quiescence.The transcription and secretion of IL-6 and TGF-β1 in ALC-treated HP-1 cells were significantly higher than those in the control group(P<0.001 between four groups).The levels of TGF-β1 synthesis were increased in the HP-1 cells that had been treated with ALC or IL-6(100ng/ml),with the highest level by 24 h,while the levels of Col1A1 and α-SMA synthesis in ALC-treated HP-1 cells were higher by 48 h.Further studies showed that ATRA not only significantly reduced the ALC-induced TGF-β1concentrations,but also decreased the ALC-induced Col1A1 and α-SMA production at 48 h.In order to know whether ATRA inhibite TGF-β1-induced phosphorylation of Smad2/3 or not,the HP-1 cells were pretreated with TGF-β1(5 ng/ml)for 45 min prior to addition of ATRA and then cultured for24 h.The results showed that ATRA could significantly down-regulate TGF-β1-induced phosphorylation of smad2/3(both P<0.001),decrease TGF-β1-induced Col1A1 and α-SMA m RNA and protein production(P<0.001 between the four groups),and increase MMP-9 m RNA and protein synthesis(both P<0.001).Conclusion: ATRA can promote the PSC transition to quiescence.ATRA can also inhibit PSC-mediated collagen synthesis and promote collagen degradation through down-regulating TGF-β1/smad2/3 signaling pathway.Thisstudy provided the experimental and theoretical basis for the prevention and treatment of pancreatic fibrosis by using ATRA.