Mechanism Of Hepatic Steatosis In Alcoholic Fatty Liver Disease Formation

Backgroud:In the middle of the last century,all of us know that fatty liver disease is induced by alcohol,and the long-term drinking can lead to alcohol liver disease,the effect for which be up to ninety-nine percents. However,the understanding and study of its mechanism focus on the metabolism of fat and the balance of energy,the understanding which focus on the reason and the way for fatty liver induced by alcohol is not clear.A large number of hepatocytes trans-differentiate into fat cells in the progress of fatty liver disease,which will seriously affect the detoxification function of the liver,then affect the function of the nerve and the kidney.This is very serious,more seriously,fatty liver may develop into alcoholic hepatitis or cirrhosis,even liver cancer.The number of people drinking increase every day,there is a great burden on individual,families and society caused by the liver diseases.We can truly find the preventive measures,and provide the experimental and theoretical basis for the treatment of alcoholic fatty liver disease,only if we get the sake of the mechanism of alcohol liver disease.Therefore,this study have practical significance.Objective:Through the experiments in vitro,there are two groups of primary culture including the primarily-cultured liver cells and the purified hepatocytes.We want to research the changes of liver cells between the control group and the experiment groups through morphology,cytochemistry and molecular biology etc,and then to study the mechanism of hepatic steatosis from hepatocytes induced by ethanol.To provide the experimental evidence of clinical drugs and the prevention for alcoholic liver disease.Methods:Primarily cultured KM mouse hepatocytes at5th day are used as experimental material in this study,and then do the following experiments:1. Primary culture of liver cells using tissue digestion with trypsin;2. Primary culture of liver cells,then get the single and highly purified hepatocytes using cell separating liquid successfully;3. There are two big experimental groups:the primary culture of the primarily-cultured liver cells(I group) and the primary culture of the purified hepatocytes(II group).Every group are divided into control group and experiment groups (50,100,200,300,400mmol/L five ethanol dose);4. Morphological observation of cell types in the primary culture of the primarily-cultured liver cells and the purified hepatocytes;5. Hepatocytes are identified by PAS glycogen staining in the two groups;6. Kupffer cells are identified by immunocytochemistry in the two groups;7. The degree of hepatic steatosis is tested by oil red O staining after the alcohol treatment for the two groups,then extract the oil red O dye with isopropanol,and determine the absorbance values at510nm with Microplate reader for quantitiative analysis;8. Crack cells with TRIzol after the alcohol treatment for the two groups,then RT-PCR and agarose gel-electronphoresis.The mRNA level changes of PPARα and PPARy are analyed quantitatively with electrophoresis by Image J analysis software;9. Crack cells with TRIzol after the alcohol treatment for the two groups,then RT-PCR and agarose gel-electronphoresis.The mRNA level changes of TNF-α and IL-6are analyed quantitatively with electrophoresis by Image J analysis software.Results: 1. Morphological observation:Under the microscope,there are several types of cell in the primary culture of the primarily-cultured liver cells;there are single and highly purified hepatocytes after separated and purified.The hepatocytes are spherical,oval or polygonal,have clear boundary、transparent cytoplasm and one nucleus or two nuclei in the middle of the cells;The volume of Kupffer cells are larger,oval or polygonal;The hepatic stellate cells are irregular,have pseudopodium;2. Identification of glycogen by PAS:Under the microscope,the cytoplasm of hepatocytes display red because of containing a large number of glycogen granules in the cytoplasm after dyed by PAS.Part of cells display red in the primarily-cultured liver cells,while almost ninety percents of cells display red in the isolated and purified hepatocytes;3. Identification of Kupffer cells by immunocytochemistry:Under the fluorescence microscope,the Kupffer cells display red because of containing a marker CD68which is the specific antigen molecule of Kupffer cells. A small proportion of cells display red in the primarily-cultured liver cells, while almost no cells display red in the isolated and purified hepatocytes;4. Identification of adipose cells:The neutral fat including triglyceride can be dyed red,because oil red O is fat-soluble dyes,can be dissolved highly in fat.Under the ethanol treatment,the cytoplasm of partial cells in the experimental groups display red after oil red O staining in the primarily-cultured liver cells, while almost no cells display red in the isolated and purified hepatocytes;5. The mRNA level changes of PPAR a and PPAR y are tested by RT-PCR,the results of the relative expressions of mRNA from the electrophoresis analysed by the Image J analysis software show:1) In the group I,the differences of the mRNA activity level of PPARa between experimental groups of different alcohol concentration and control group have statistical significant(P<0.05),and the mRNA activity level have a trend of lower with the alcohol concentration increased;The differences of the mRNA activity level of PPARy between experimental groups of different alcohol concentration and control group have statistical significant(P<0.05),and the mRNA activity level have a trend of increase with the alcohol concentration increased;2) In the group Ⅱ,the result show the differences have no statistical significant(P>0.05).6. The mRNA level changes of immune factors(TNF-a and IL-6) are tested by RT-PCR,the results of the relative expressions of mRNA from the electrophoresis analysed by the Image J analysis software show:1) In the group I,the differences of the mRNA activity levels of TNF-α and IL-6between experimental groups of different alcohol concentration and control group have statistical significant(P<0.05),and the mRNA activity levels have a trend of increase with the alcohol concentration increased;2) In the group II,the result show the differences have no statistical significant(P>0.05).Conclusion:1. Primarily cultured KM mouse hepatocytes at5th day are used as experimental material in this study,this is the useful material to get the liver cells;2. In this study,primary cultures of liver cells use the method of tissue digestion with trypsin and adherent culture in vitro,then use cell separating liquid and appropriate conditions of centrifugation to get the single and highly purified hepatocytes successfully;3. The adipose cells are identified by oil red O staining in this study,and analysis quantitiatively the degree of steatosis through the absorbance values at510nm determined by Microplate reader after extract the oil red O dye with isopropanol;4. Compared with isolated and purified hepatocytes, the primarily-cultured liver cells undergo hepatic steatosis after the same ethanol treatment, while the purified hepatocytes fail to form lipid cells under the same ethanol treatment.The mRNA level of PPARa decrease,and PPARy,TNF-α，IL-6improve obviously compared with control group;5. Purified hepatocytes lose the ability to trans-differentiate into fat cells; It is possible that the transdiferentiation of hepatocytes into fat cells is because of non-hepatocyte cells are able to provide some factors including immune factors upon ethanol stimulation.