Study On The Serum Markers Screening Of Cirrhosis Of The Liver Tissue Of Chronic Hepatitis B

The research background and purpose:Liver fibrosis is abnormal connective tissue hyperplasia and excessive accumulation of extracellular matrix diffuse intrahepatic pathological process. Hepatitis virus can cause liver inflammation and then activate the immune systems to repair liver tissue. If the repair process is excessive or out of control, the liver function and structure were changed and then the liver fibrosis is formed. Slight change for liver fibrosis,Serious changes cause the structure of hepatic lobule happens disorder, gradually form false flocculus, eventually evolved into liver cirrhosis.a lot of clinical research results show that [1], the body oneself internal system can degradate fiber,the system can realize reversal of liver fibrosis and Early liver cirrhosis also had been reversed[2][3], and decompensated cirrhosis by liver tissue badly damaged cannot be reversed.Therefore, The early diagnosis and treatment of liver fibrosis can prevent liver fibrosis from developing to liver cirrhosis.It has very important significance to improve the prognosis of hepatopath.Early serum markers of hepatic fibrosis mainly reflect the dynamic changes of the liver collagen synthesis and catabolism, cannot really reflect the degree of collagen deposition. Currently the "gold standard" accepted by the diagnosis of liver fibrosis is liver biopsy pathology examination. The limited clinical ap plication is because it’s traumatic and limitations, and patients suffer from pain, less likely to be repeated inspection. The imaging examination of liver fibrosis is more reliable for the severe phase, but the lack of specificity for mild and moderate phases.Currently there is lack of a useful serum marker of clinical evaluation of liver fibrosis degree[4]. so, finding out the serum marker of the early diagnosis of liver fibrosis, the assessment of liver fibrosis degree to guide clinical diagnosis and treatment is an important research topic in the current medicine.Metabolomics(metabonomics/metabolomics) is a science mainly researching the types, quantities and the change rule of metabolites produced by body’s different disturbances. Metabonomics had been applied to detect and find early serum markers of metabolites in liver failure[5], nonalcoholic fatty liver[6], liver cancer[7][8], liver damage caused by the viral factors such as drugs and poisons[9][10],thus provides a new method for the early diagnosis and treatment of these diseases.This experiment adopts high performance liquid chromatography(HPLC)- mass rail ion trap mass spectrometry(UPLC/LTQ Orbitrap- MS) technology to separate and extract serum metabolites of different fibrosis phase including chronic hepatitis B, compensated and decompensated liver cirrhosis in order to discover the most different metabolites as serum early markers of liver fibrosis.Methods:1.Randomly choiced 34 patients with chronic hepatitis B in October 2011-September 2013 in yantai city infectious diseases hospital, all patients underwent liver biopsy pathologic examination, randomly selected 15 patients with decompensated cirrhosis hospitalized from 6 people’s hospital in dalian. 20 cases of healthy volunteers as the normal control group, liver function is regular, etiology examination negative. All the groups of people return on an empty stomach blood 4 ml on the premise of informed consent, then separate and return serum within half an hour,- 80℃ cryopreservation.2.High performance liquid chromatography(HPLC)- rail ion trap mass spectrometry mass spectrometry(UPLC/LTQ Orbitrap- MS) technology to detect the histologic diagnosis of chronic hepatitis B, compensatory phase cirrhosis of the liver, in patients with decompensated cirrhosis serum metabolites, find metabolite markers of liver fibrosis.2.1 Sample preparation: The sera were thawed at room temperature before analysis. 400 ul methanol protein was added to 100 ul serum, vortex 60 seconds, centrifuged at 13000×g for 15 minutes at 4 ℃. the supernatant fluid(400 ul) was lyophilized.2.2 Ultra high performance liquid chromatography(HPLC) analysis: using ultra high performance liquid chromatography(HPLC) analyzer. Wash column using 10 cm x 1.7 x 2.1 mm um C18 column according to the gradient change with the different wash, acetonitrile solution according to the material length of stay in the column, complete separation of various kinds of metabolites.2.3 High performance liquid chromatography(HPLC)- rail ion trap mass spectrometry mass spectrometry(UPLC/LTQ Orbitrap- MS) : USES the LTQ Orbitrap mass spectrum analyzer, to ESI + model has separated the metabolites of operation.2.4 Data processing and statistical analysis: through the Micromass Marker Lynx for every peak retention time(tr) and m/z of data, standardization output after each peak ion density, principal component analysis(PCA). According to the test results, looking for differences in larger metabolites. For the statistical analysis of SPSS. 18.2.5 Determine the metabolic product name: name according to accurate to determine the molecular weight metabolites.Results:1.Of 34 patients with chronic hepatitis B liver biopsy pathologic examination, the results showed: S1:8 cases,S2: 5 cases, S3:1 case, S4:20 cases. 14 cases of S1- S3 period, for chronic hepatitis B group; 20 patients of S4 period with early cirrhosis compensatory phase cirrhosis namely group.2. In the normal control group, chronic hepatitis B group, compensatory phase cirrhosis group, patients with decompensated cirrhosis group serum spectrum found in the comparison of the metabolism, relatively normal control group, chronic hepatitis B, compensatory phase cirrhosis of the liver, in patients with decompensated cirrhosis methionine showed a trend of gradual decline, coenzyme Q1, carboxyethyl arginine, gump ammonia deoxycholic acid showed a trend of rising.Conclusion:Methionine(Methionine), coenzyme Q1(coenzyme Q1), carboxyethyl arginine(octopine), gump ammonia deoxycholic acid(GDCA) can be used as a metabolic markers of hepatic fibrosis.