The Establishment Of Method For Detection Of Cytoglobin And Its Application In Rat Liver Fibrosis Model

There are more than5%of the population suffer from diseases associated with of liver cirrhosis, in many countries, liver cirrhosis has become a more common cause of death. With the development of cell biology and biochemistry, that liver fibrosis is a chronic liver injury in a variety of cells in close contact, and through a variety of cytokines and extracellular matrix proteins influence each component networks to regulation. Great harm liver fibrosis, structural damage due to liver tissue, resulting in the portal venous system vascular resistance increases, the formation of portal hypertension, leading to splenomegaly, ascites and esophageal and gastric varices, variceal upper gastrointestinal bleeding potential danger. And because of liver fibrosis between the normal liver cells due to blood microcirculation channel deposition of fibrous tissue components caused by circulatory disorders affecting the blood supply to the liver cells, so that damage due to inflammation of the liver cell damage difficult to repair or even increased, until function less and less of normal liver cells, leading to cirrhosis, and even the development of liver cancer. For the treatment of liver fibrosis mainly from the starting points:(1) remove the cause treatment. Severe liver injury site will usually produce fibrosis, and inflammation and injury severity was positively correlated with liver fibrosis, if early step in removing the cause, such as viruses, alcohol and poisons, which is the most effective treatment for cirrhosis prevention measures.(2) inhibition of hepatic stellate cell activation and proliferation.(3) increased hepatic extracellular matrix (extra cellular matrix, ECM) degradation. Such as matrix metalloproteinases and their inhibitors TIMP, or use of urokinase-type plasminogen activator and inhibitor.(4) Enhanced regeneration. Such as hepatocyte growth factor (HGF) can not only stimulate the liver to regenerate, but also to prevent the occurrence of liver fibrosis and accelerate their recovery.(5) stem cell therapy. Studies have confirmed that mesenchymal stem cells (MSC) can be played by inducing apoptosis of HSC reduce liver fibrosis. Although these drugs effective in the treatment of cirrhosis of the process, but the side effects are large, such as hepatocyte growth factor has caused cancer risk, and urokinase-type plasminogen activator and its inhibitors may cause bleeding or thrombosis.The diagnosis of liver fibrosis are in three ways:First, biopsy diagnosis of liver which is still the "gold" standard, as an invasive examination, has obvious limitations, has certain risks, patients not easy to accept, it is difficult to ensure a drawn picture of the liver can react with some false negative. Two serological diagnosis:primary liver fibrosis four (PC Ⅲ、LN、HA、Ⅳ-C), matrix metalloproteinases (MMP2) and its inhibitor (TIMP1) and cytokines checks, but the lack of specificity of the liver. Third, imaging diagnosis:The main use of B-ultrasound, CT and MRI to be checked. Although not directly diagnosis of hepatic fibrosis, but there is still a certain value. Also based on biochemical and ultrasound detection of liver elasticity Fibrotest and Fibroscan technology, non-invasive, rapid, quantitative estimates of the degree of liver fibrosis, but can not accurately distinguish between adjacent fibrosis stage, it still can not completely replace liver biopsy pathology checks. Therefore need to find a new approach to the diagnosis of hepatic fibrosis or indicator.Now that hepatic stellate cells (hepatic stellate cell, HSC) in the development of liver fibrosis leading role. In a variety of pathogenic factors, is activated and quiescent HSC proliferation, transformation into myofibroblast-like mother cells synthesize and secrete ECM increases, both the synthesis and release large amounts of matrix metalloproteinase inhibitors (TIMPs), the interstitial collagen Protease enzyme activity decreased, ECM decreased degradation, resulting in the accumulation of ECM and cirrhosis of liver fibrosis as well. Given HSC in the pathogenesis of hepatic fibrosis in the leading role, the majority of studies in liver fibrosis HSC as target. Kawada, etc. During Proteomics rat stellate cells, they found a new protein stellate cellactivation-assosiated protein (STAP). The protein in hepatic stellate cell activation process significantly upregulated, and only in hepatic stellate cells, their biochemical characteristics indicate that it is an endogenous peroxidase, can break down over hydrogen peroxide and lipid peroxides, the latter two reportedly can activate hepatic stellate cells, promote liver fibrosis. By homologous comparison, in the human liver and found in the genome of human hepatic stellate cell activation-associated protein (human Stellate cell activation-associated protein hSTAP), the nucleotide sequence of murine hepatic stellate cell activation-associated protein (rat Stellate cell activation-associated protein rSTAP)97%nucleotide sequence homology with the gene (GenBank AB057769) is located on chromosome17, encoding190amino acid residues. STAP is currently in the international arena has also been named cytoglobin (Cygb) or histoglobin, it belongs to a newly discovered hexacoordinate globin superfamily. Chinese scholars Ruian Xu, etc.(2006) constructed a recombinant rat Cygb adeno-associated virus expression vectors to gene therapy approach, research on liver fibrosis in rats Cygb therapeutic effect, the findings show that in the rat stellate cells overexpressing rCygb can alleviate oxidative stress, inhibition of fibroblast-like female muscle cell differentiation, while reducing extracellular matrix deposition. Hong Kong scholar Man KN (2008) demonstrated in animal experiments in mice Cygb in CCl4induced liver fibrosis increased expression and may act as an early marker of liver fibrosis.Based on this we designed the following experiment, through the establishment of testing methods Cygb liver fibrosis model Cygb content changes, explore its potential as indicators of the likelihood of detection of liver fibrosisCygb testing we are creating a double-antibody sandwich ELISA. The use of the constructed recombinant human engineered bacteria (recombinant human Cytoglobin) rhCygb, purified by SDS-PAGE recycling, re-test the purity by SDS-PAGE and Western-blot for identification. Monoclonal antibodies for laboratory previously prepared and used HRP-labeled. Double-antibody sandwich ELISA method requires pairing antibody screening, screening out the highest OD value pair. And make a lot of preparation, purification, and then through orthogonal test antibody-coated ELISA antibody concentration and dilution to the purified antigen rhCygb do calibration standard curve, linear range, accuracy, sensitivity evaluation ELISA method.Liver fibrosis model construction we use SD male rats were injected CC14method by injecting different concentrations of CC14were constructed with mild, moderate and severe liver fibrosis model by detecting alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver fibrosis four (LN, HA, P Ⅲ NP, Ⅳ-C) and a biopsy to determine model. Finally detection of serum liver fibrosis Cygb content, data processing using spss13.0.The results show the purity of rhCygb after purification is more than95%, can be used as calibration antigens. rhCygb by protein G purified monoclonal antibodies,90%purity, HRP labeled rate of50%-60%. Paired experiments show to5①as coating antibody,2③the best combination for the detection of antibodies, orthogonal OK to2μg/ml coated, enzyme labeled antibody1/1600dilution for the optimum condition, in order to establish a standard curve to rhCygb values of concentrations as abscissa and ordinate corresponding absorbance values plotted working curve, curve equation was obtained Y=0.3395X-0.1695, at0-1500ng a good linear correlation coefficient R2is99.41%, the detection limit was8.7ng. Rat liver fibrosis model was successfully constructed biopsy revealed different concentrations of CC14to induce varying degrees of fibrosis, the Knodell index score shows the model group were mild, moderate and severe liver fibrosis. Model group and the normal group indexes (alanine aminotransferase, aspartate aminotransferase and liver fibrosis four) There was significant difference (P<0.05), but moderate and severe model model group between groups laminin (LN), hyaluronic acid (HA),Ⅲprocollagen (P III NP) no significant difference between the groups of each model type Ⅳ collagen (Ⅳ-C) no significant difference. Laminin (LN), hyaluronic acid (HA),Ⅲ procollagen (P Ⅲ NP) and liver fibrosis correlation coefficients were0.66,0.826,0.741, P values were less than0.01. The type IV collagen (IV-C) and no correlation of liver fibrosis. Normal serum samples Cygb content of36.11±5.01ng/ml, mild levels of serum sample cell globin content of119.11±13.00ng/ml, moderate levels of serum sample cell globin content of161.24±14.46ng/ml, severe levels of serum sample cell globin content of203.67±29.33ng/ml. Pairwise comparisons between the groups had significant difference (P <0.01), and liver cells globin content fibrosis correlation coefficient is0.906.