The Impact Of HepG2.2.15 Cell Steatosis On HBV Degeneration And SOCS-3 And SREBP-1c Pathway

Background Chronic Hepatitis B(CHB) is caused by hepatitis B virus infection, it has becamea serious global public health problem. About 20 million people have been infected with HBVworldwide, of which 350 million people are chronic HBV infection, about 15% to 25% of themeventually died of liver failure , cirrhosis and primary hepatocellular carcinoma ( HCC) relatedto HBV infection. The 5-year mortality of chronic hepatitis B, compensated and decompensatedcirrhosis was 0% to 2%, 14% to 20% and 70% to 86%. Over the years, the incidence rates ofhepatitis B in china were in the forefront , and were seriously damage to people's health. Thehepatitis B surface antigen carrier rate among 1 to 59 years old people in china was 7.18%.( byMinistry of Health,2006).Non-alcoholic fatty liver disease (NAFLD) is one of the liver injury acquired frommetabolic stress, characterized by diffused macrovesicular fatty liver degeneration except otherfactors that can caused liver damage, such as excessive alcohol consumption and otherspecifically factors. NAFLD is one of a metabolic stress liver injury closely related to insulinresistance(IR)and genetic susceptibility. Disease spectrum including non-alcoholic fatty liver(NAFL),non-alcoholic steatohepatitis (NASH) and cirrhosis related to NASH.. Epidemiologicalsurveys showed that fatty liver disease has been prevalent in the world. About 20 to 30% or morepeople are suffered from NAFLD. In china the prevalence of NAFLD in the past 10 years hasbeen doubled, NAFLD can damage the liver directly , and even can cause acute liverfailure(ALF).It have been found by clinical study that many CHB patients are associated with NAFLD,this phenomenon have been valued by more and more of scholars. Comprehensive analysis theresults of a number of large-scale epidemiological survey, the incidence rate of CHB Associatedwith NAFLD is about 11 to 15% in the population. It has been confirmed that, up to 62% ofhepatitis C patients associated with NAFLD , The IR in the Hepatitis C virus infected people iswidespread. HCV genotype 1 and 4 can cause hepatic steatosis through the IR, and genotype 33can cause hepatic steatosis directly. These metabolic factors accelerated the process of liverfibrosis and increase the risk of hepatic carcinoma. Whether the HBV can lead to IR or not as theHCV? Can steatosis impact the HBV replication and antigen synthesis ? Is the HBV paly a promote or retard function in the course of hepatic steatosis ? These problems have been greatlyconcerned, and they are also the main content of our research.The study can be divided into the following four parts:Part 1 The study of establishing the HepG2.2.15 cell steatosis model.Part 2 The impact of HepG2.2.15 cell steatosis on HBVDNA degeneration and antigensecretion.Part 3 The impact of HepG2.2.15 cell steatosis on the SOCS-3 and SREBP-1c expression.Part 4 The impact of the transforming growth factorβ1(TGFβ1) on the HepG2.2.15 cellsteatosis.ParPart 1 The study ofestablishing the HepG2.2.15 cell steatosis modelExperiment 1 : study ofcell culture about HepG2 and HepG2.2.15 cellsObjective To establish HepG2/HepG2.2.15 cryopreservation, recovery and passageMethods and conditions, master the cell proliferation characteristics and the replication andprotein synthesis of HBV.Methods Correct methods of sterile culture was used , G418 was regularly added to thecell culture medium, the cell growth conditions was observed by inverted microscope ,cellcounting and Dynamic identification was done by trypan blue staining methods , HBV DNA inthe supernatant was detected by real-time fluorescence quantitative methods, HBsAg andHBeAg were detected by time-resolved fluorescence analyzer.Results Cells exponential growth can be maintained by cell culture with 10% of FBS in it ,the 90% survival rate of cell recovery can be guaranteed by frozen solution with 70% FBS init .HBVDNA, HBsAg and HBeAg in culture supernatant increased steadily along with theculture time.Conclusion HepG2/HepG2.2.15 growed fast, a higher serum concentration culture andcryopreservation solution were required. Regularly added G418 solution to the HepG2.2.15cellculture medium can guarantee the bility of HBV replication and antigen secretion. ExperimenExperiment 2: The study ofestablishing the HepG2.2.15 cell steatosis modelObjective To establish aboratory conditions of hepG2/HepG2.2.15 cell steatosisMethods The Oleic acid (OA) was celected to be the steatosis inducer and 0.1 to 0.4 mMconcentration were setted to to induce HepG2/HepG2.2.15 cells steatosis. Cells were dividedinto control group, DMSO group, and OA group. The relative activity of cells were detected byMTT method, and producted the cell growth curve according to the MTT result. The Lipiddroplets within the the cells were stained by Oil red O-hematoxylin staining method .Results The cells growth curve showed that the cell proliferation keep activity, when theconcentration of OA≤0.2mM, and cells proliferation could decreased gradually if theconcentrations of OA> 0.2mM, and the DMSO had no effect on the cell proliferation .The oilred O staining results showed that , in the 0.2mM, orange small lipid droplets in the cells beganto appear ed at 24h, these lipid droplets were gradually increased,and bagan biger slowly ,andthe cell steatosis could be formed at 72h.Conclusion HepG2/HepG2.2.15 cells significant steatosis can be formed at 72h,undedr the role of 0.2mM OA without affecting the cell proliferation .The degree of steatosis ofHepG2.2.15 cells are lower than HepG2 cells. There were significant difference betweensteatosis rates of HepG2 and HepG2.2.15 cells at 24h, 48h.and was't at 72h.Experiment 3: The impact ofHepG2/HepG2.2.15 cell steatosis on TG and ALTObjective To xplore the impact of HepG2/HepG2.2.15 cell steatosis on TG and ALTMethods The cells were divided into HepG2 cell control group (C1 group) and HepG2 cellfatty group (F1 group), HepG2.2.15 cell control group (C2 group) and HepG2.2.15 cell fattygroup (F2 group). according to the methods established in Experiment 2 of part 1. Cells werelysed by repeated freeze-thaw method , TG intracellular and ALT in the supernatant weredeteced by Automatic biochemical analyzer.Results1. TG Paired t test results showed that : TG of F1 and F2 group in 24h,48h and 72h weresignificantly higher than C1 and C2 group(P＜0.05), Independent sample t test showed that. TGof F1 group were significantly higher than F2group. 2. ALT Paired t test results showed that : ALT of F1 and F2 group in 24h,48h and 72h weresignificantly higher than C1 and C2 group(P＜0.05), Independent sample t test showed that. ALTof F1 group were significantly lower than F2 group.Conclusion HBV could inhibit the TG produce,and increase Cell membrane damagePart 2 The impact of HepG2.2.15 cellsteatosis on HBVDNA degeneration and antigen secretionExperiment 1 The impact ofHepG2.2.15 cell steatosis on HBVDNAObjective To explore the impact of HepG2.2.15 cell steatosis on HBV replicationMethods The cells were divided into control group (C2 group) and fatty group (F2group), according to the methods established in experiment 2 of part 1. HBV DNA in thesupernatant was detected by real-time fluorescence PCR quantitative methods.Results The HBVDNA (log) rising curve in the control group was one straight line. andin fatty group was one tortuous line. Paired t test results showed that : there was't statisticallysignificant. Between the HBVDNA of F2 group and C2 group in 24h, 48h, 72h (t =- 2.93,P=0.784;t =0.065, P=0.951;t =1.551, P=0.182)Conclusion HepG2.2.15 cells Steatosis had no significant affect on replication capacityof HBV.Experiment 2 The impact ofHepG2.2.15 cell steatosis on HBsAg and HBeAgObjective To explore the impact on secretion of HBV antigen markers of HepG2.2.15cell steatosisMethods The cells were divided into HepG2.2.15 cell control group (C2 group) andHepG2.2.15 cell fatty group (F2 group), according to the methods established in Experiment 2of part 1. HBsAg and HBeAg in the supernatant were detected by time-resolved fluorescenceanalyzer.Results1.HBsAg: Paired t test results showed that : HBsAg of F2 group in 24h,48h and 72h weresignificantly lower than C2 group(t=32.985, P＜0.001; t=5.843, P=0.002; t=10.517,P＜0.001),HBsAg in steatosis HepG2.2.15 cells were significantly decreased . 2.HBeAg:Paired t test results showed that there were not statistically significant betweenthe HBsAg of F2 and C2 groups( t=0.283, P=0.789; t=0.523, P=0.623; t=-0.542, P=0.611) .Conclusion There are different impact on HBsAg and HBeAg of HepG2.2.15 cellsteatosis. It Could significantly inhibit the synthesis of HBsAg and had no impact on HBeAg.The decrease of HBsAg was related to the changes of the cell membrane structure.Part 3 The impact of HepG2.2.15 cellsteatosis on the SOCS-3 and SREBP-1c expressionObjective To explore the impact on SOCS-3 and SREBP-1c of HepG2.2.15 cell steatosisMethods The cells were divided into HepG2 cell control group (C2 group) and HepG2cell fatty group (F2 group), HepG2.2.15 cell control group (C2 group) and HepG2.2.15 cellfatty group (F2 group). According to the methods established in Experiment 2 of part 1.SOCS-3and SREBP-1 protein expression in cells were deceted by immunohistochemistry, SOCS-3 andSREBP-1mRNA were deceted by real-time PCR methods.Results1. The immunohistochemistry results showed that SOCS-3 staining of HepG2 andHepG2.2.15 cells gradually fades and SREBP-1c staining gradually deepened along withtreatment the treatment time.2. The SOCS-3 mRNA : the SOCS-3 mRNA of C2 group was far below the C1 group (P<0.001), F1 group was significantly lower than the C1 group (P = 0.003), F2 group was lowerthan C2 group, but it had no statistically significant (P = 0.173). there was interaction betweencell *fatty factors (F=25.547,P＜0.001).3. The SREBP-1c mRNA:the SREBP-1c mRNA of C2 group was higher than C1 group(P=0.013), the SREBP-1c mRNA of F1 and F2 group were all increased compared with C1 andC2group, there was significant difference between F2 group and C2 group(P=0.002), and therewas't significant difference between F1 and C1 group(P=1.000). There was interactionbetween cell*fatty factors(F=5.04,P＜0.03).Conclusion1. SOCS-3: The HBV DNA could down regulate the expression of SOCS-3 mRNA. theSOCS-3 protein and mRNA expression of HepG2/HepG2.2.15 cell steatosis were all downwardregulated, and the regulated degree of HepG2 cell was higher than HepG2.2.15 cell.2. SREBP-1c:The HBV DNA could upward regulate the expression of SREBP-1c mRNA.The SREBP-1c of the HepG2/HepG2.2.15 cell steatosis were all upward regulated ,and and the regulate degree of HepG2.2.15 cell was higher than HepG2 cell.Part 4 The impact of the transforminggrowth factor (TGFβ1)on the HepG2.2.15 cell steatosis.Objective1. To explore the impact of TGFβ1 on HepG2.2.15 cell steatosis.2. To explore the relationship between TGFβ1 and SOCS-3 mRNA/SREBP-1c mRNA inthe HepG2/HepG2.2.15 cell steatosis process methods.Methods The cells were divided into HepG2/HepG2.2.15 cell control group (C1/C2group) and HepG2/HepG2.2.15 cell fatty group (F1/F2 group). Add 5ng/ml TGFβ1 into the twocells culture systems, then it formed another two groups: TGFβ1 intervention group(T1/T2group) and the fatty + TGFβ1 intervention group (FT1/FT2 group). HBsAg and HBeAg in thesupernatant were detected by time-resolved fluorescence analyzer. HBV DNA,SOCS-3 mRNAand SREBP-1 mRNA were deceted by real-time PCR.Results1. HBVDNA:There was't significant difference between the HBVDNA of T2 andC2group, TF2 and F2 groups (P=0.056,P=0.178). there was't interaction between fatty *fattyfactors(F=10.026,P= 0.872).2.HBsAg:The HBsAg of T2 and TF2 groups were less than C1 and F2 groups, there wassignificant difference between TF2 and F2 groups(P＜0.001), and there was't significantdifference between T2 and C2 groups(P=0.961). There was interaction between fatty*TGFβ1factors(F=15.49,P= 0.001).3. HBeAg:The HBeAg of T2 and TF2 groups were less than C1 and F2 groups, therewere significant difference between them(P=0.004,P=0.034), There was't interaction betweenfatty*TGFβ1 factors(F=0.265,P=0.813).4. SOCS-3 mRNA:The SOCS-3 mRNA of T1,TF1,T2 and TF2 groups were respectivelylower than C1,F1,C2and F2 groups, there was significant difference between them(P=0.050, P＜0.001, P＜0.001, P＜0.001). There was interaction between fatty*TGFβ1 factors(F=1.872,P=0.184). There was't interaction between cell type and TGFβ1 factors(F=12.628,P=0.001).5. SREBP-1c mRNA: The SREBP-1c mRNA of T1,TF1,T2 and TF2 groups wererespectively higher than C1,F1,C2 and F2 groups, there were significant difference betweenthem(P≤0.001). there was significant difference between TF2 and TF1(P＜0.001).There was't interaction between fatty*TGFβ1 ,cell type*TGFβ1 factors(F=7.655,P=0.009；F=9.70,P=0.003).Conclusion1. There was't impact on HBV duplication of HepG2.2.15 cell steatosis.2. The TGFβ1 could inhibit the Expression of HBsAg, and the inhibitory ability wasinfluenced by the cell steatosis , the inhibitory ability on steatosis group was stronger thannon-steatosis group.3. The TGFβ1 could inhibit the expression of HBeAg, and the inhibitory ability was'tinfluenced by the steatosis factor.4. The TGFβ1 could down regulate the expression of SOCS-3 mRNA, and this inhibitoryability was influenced by steatosis factor,and have no relationship with the cell type(that thepresence of HBV).5. The TGFβ1 could up regulate the expression of SREBP-1c mRNA, and this inhibitoryability was't influenced by steatosis and cell type factors.