Study Of Serum TNF-a And Hepatocyte Apoptosis In Alcoholic Liver Disease Rat

IntroductionThe occurrence of Alcoholic Liver Disease ( ALD ) is related with many factors. These are genetic factor, immune factor, oxidation and hypoxia. Among these: more and more studies focus are the role of cytokines in alcoholic liver disease. Cytokines take part in the inflammation of liver cell and induce hepato-cyte apoptosis. Tumor Necrosis Factor - (TNF - ) plays important role in the progress of alcoholic liver disease. In this study we try to demonstrate the relationship among serum TNF - a level. Apoptosis of liver cell and the dosage of alcohol fed in rat alcoholic liver disease animal model. We found that liver damage is related with alcohol dosage.Experiment materials and Methods1. Experiment objects;35 male Wistar Rats, weight 280 -300g.2. Experiment material and instruments:TUNEL Kit, Protease K, Clean table, Light microscope, Electronic balance, All kinds of alcohol, TBS\PBS buffer, Beaker, dropper, Straws, 10% formaldehyde solution, Dimethylbenzene. TNF RIA Kit3. Experiment procedures:( 1) Establish animal model: Randomize 35 rats into 4 groups, one is control , the others are experiment groups. Control group is fed water, experiment groups are fed 4g. kg-1M-1,6g. kg-1M-1,8g. kg-1M-1of 40 percent alcohol re-spectively for two months.(2) After anaesthesia, we exposed external jugular vein and took 2 ml blood . Serum was separated and stored at 200 C: At once, we cut open thoracic cavity; abdominal cavity; exposure heart; shears open pericardium, left ventricle; insert catheter and fixation, at the same time, we use surgical scissors to cut open right auricle to put blood, then , use PBS buffer from left ventricle to right auricle, via systemic circulation. After liver color turn bright red into grey white, we stop filling. Stabilize liver, section 10 leaves for sample, 5 for paraffin embedding, the other for HE staining.( 3) Detect level of TNF - a and ALT in serum.(4)Apoptosis was detected by terminal deoxynucleotidyl trasferase mediated dUTP nick end - labeling technique ( TUNEL test). We treat sample by 3% H202and wash it by distilled water , use protease K to digest the sample, wash it by distilled water, add tagging buffer to the sample, wash it by TBS, add blocking fluid and biotic anti - DIG - antibody, reach for 30 minutes, wash it by TBS. We add SABC for 30 minutes reaction, wash it by TBS, stain it by DAB.(5)Result determination standard: TUNEL expression: We also used ap-optosis index ( AI) to evaluate semiquantitatively the immunoexpression, judging the cases with brown nuclei staining as positive. We counted 200 cell in 1 - 3 high time positive field at random, Calculation formula; AI = apoptosis cell/ cell sum x 100%.4. Statistical analysis:Database was set up in SPSS for windows 10.0. ANOVA and Pearson correlation analysis was used to solve the data.Result1. Level of TNF - a and ALT in serum:Level of TNF - a in serum is evaluated with the increasing dosage of alcohol. So did the level of ALT.2. Detection of hepatocyte apoptosis:Apoptotic cell was Occasionally detected in control group, and was more detected in experiment groups. There is significant positive connection between dosage of alcohol and number of apoptotic cell. The more dosage of alcohol was, the more number of apoptoic cells was.3. Relationship between level of TNF - a and hepato cellular apoptodid in dex ;Level of TNF - a and hepatocellular apoptosis index is evaluated with the increasing dosage of alcohol. The correlation between TNF and hepatocellular apoptosis was positive.DiscussionTNF - a is mainly produced in macrophage and monocyte. It can induce cell apoptosis in liver. TNF - a is mainly produced in activated kupffer cell and takes part in the occurrence and progress of ALD. TNF - a increases in ALD patient ' s serum and is related with biochemical parameter reflecting liver damage.Our result showed that the level of TNF - aand ALT in serum was elevated with the increasing dosage of alcohol fed rat. HE staining of liver se...