The Relationship Between TGFβ1 And TNF-α With Chronic Hepatitis B With Non-alcoholic Fatty Liver Disease

The infection of HBV is a serious public health problem. And along with the economic level enhancement, fatty liver disease is becoming more and more common. In recent years, the Nonalcoholic fatty liver disease (NAFLD) increased year by year, resulting in liver steatosis and chronic hepatitis B (CHB) in combination in the same cases of individuals increased significantly. But the relationship between the chronic hepatitis B and fatty liver are reported less at home and abroad, and therefore it draws many scholars'attention. As well as we known, some chronic liver disease such as chronic hepatitis B, hepatitis C virus infection, hepatocellular carcinoma (HCC) and NAFLD are related to the immune state. Therefore, this research begin with the two cytokines which has close linked to NAFLD and chronic hepatitis B, to discuss the relationship between the two cytokines and chronic hepatitis B with NAFLD.Recent research found that the expression of transforming growth factorβ1(TGFβ1) and its receptor in nonalcoholic fatty liver tissue is increased. And the tumor necrosis fator Altha (TNF-a) play an important role in liver lipid metabolism disorders. Compare the change of serum TGFβ1 and TNF-a in CHB patients with NAFLD or not, and analysis the function of TGFβ1 and TNF-a in the patients with both CHB and NAFLD will provide new evidence for clinical diagnosis and treatment for such disease.The thesis includes two parts. PartⅠ:Clinical experiments. Observation the level of serum TNF-a and TGFβ1 in the patients of CHB with NAFLD. PartⅡ:cell experiments. This part includes two experiments:1 the establishment of hepG2.2.15 cell steatosis models induced by oleic acid; 2 the effect of TGFβ1 on hepG2.2.15 cell steatosis. PartⅠ:Clinical experiments Observation the level of serum TGFβ1 and TNF-a in the patients Of chronic hepatitis B with non-alcoholic fatty liver diseaseObjective:Observation the level of serum TGFβ1 and TNF-a in the patients of CHB with non-alcoholic fatty liver disease.Methods:Collect CHB with NAFLD patients and CHB patients'cases, ELISA assay to test the serum TGFβ1 and TNF-a in these patients. The patients with NAFLD were divided into three groups,include mild,moderately,severe NAFLD, according to Liver and Spleen CT ratio by Abdominal CT.Results:The serum concentrations of TGFβ1 and TNF-a in CHB merger NAFLD group is significantly higher than the control group (P<0.001 and P<0.001). The serum concentrations of TGFβ1, TNF-a increased with the severity of NAFLD gradually. And there exists combined correlation between the serum TGFβ1 and TNF-a level.Conclusion:①TGFβ1, TNF-a has clinical value in the pathogenesis of CHB combined with NAFLD.②n the patients with both CHB and NAFLD, the level of serum TGFβ1 and TNF-a has a close relationship with the degree of fatty liver disease.PartⅡ:Cell experimentsExperiment I:The establishment of hepG2.2.15 cellsteatosis model induced by Oleic acidObjective:To establish cells models of the CHB complicated with NAFLD.Methods:The cells model was established by treating human hepatocarcinoma cell stain HepG2.2.15 with different concentrations of oleic acids respectively. The optimum concentration of oleic acids should be definited by MTT colorimetry test. After the cells were treated for some time, the fatty droplets in cells could be observed by Oleic red O-hematoxylin staining.Results:After treating HepG2.2.15 cells with 0.05mM,0.10mM,0.2mM,0.3mM,0.4mM oleic acid for 24h,48,72h, all led to disturbance of lipids metabolism, obvious triacylglycerol accumulation in cells under light microscope by Oleic red O-hematoxylin staining, and dramatically increased red fatty droplets compared with control group; The content of red fatty droplets increased when the concentrations of oleic acid were raised. After treating HepG2.2.15 cells with 0.2mM oleic acid for 72h, human hepatocarcinoma cell stain HepG2.2.15 has the reddest fatty droplets in cell.Conclusions:We could succeed to establish cells model of the CHB complicated with NAFLD by treating HepG2.2.15 cells with 0.2mM oleic acid for 72h. Objective:To observe and study the effect of TGFβ1 on hepatocarcinoma cell HepG2.2.15 steatosis and if they could aggravate the hepatocyte steatosis by establishing hepatocyte infected by hepatitis B virus steatosis cells model.Methods:The cells were divided into three groups:the first is only plus oleic acid group (0.2mM); the second is plus oleic acid (0.2mM) and TGFβ1 group, the concentration of TGFβ1 was 10ng/ml; the third is control group which was neither plus oleic acid nor TGF-β1. After the treatment with oleic acid plus TGFβ1 for 72h, the fatty droplets in cells could be observed by Oleic red O-hematoxylin staining; the content of intracellular triglyceride was detected by Automatic Biochemical analyzer after repeated freeze-thaw cells method; the level of ALT in extracellular medium was detected by Automatic Biochemical analyzer.Results:①we found that the red fatty droplets have increased in oleic acid plus TGFβ1 group compared with oleic acid alone group in light microscope by Oleic red O-hematoxylin staining;②the content of triglyceride in oleic acid plus TGFβ1 group was significantly higher than in oleic acid alone group (P<0.001), oleic acid alone group was significantly higher than that in normal control group (P<0.001);③the level of ALT in extracellular medium in oleic acid plus TGFβ1 group was significantly higher than in oleic acid alone group (P< 0.001), the oleic acid alone group was higher than that in normal control group (P< 0.001).Conclusions:TGFβ1 can make hepatocyte cell models induced by oleic acid respectively bad, such as obvious increased intracellular triacylglycerol accumulation in cells and dramatically increased leakage of ALT, and thus aggravate HepG2.2.15 cell steatosis.