Circulating MiR-185, An Important Clinical Diagnosis Role For Hepatic Fibrosis

Background:Chronic liver disease caused by viral hepatitis, alcoholism, schistosomiasis infection is most common in China. The important pathological chronic liver disease (mainly chronic viral hepatitis) is based on liver fibrosis. liver fibrosis further development of cirrhosis or liver cancer, the mortality of patients with high quality of life is also quite poor. Currently, Western medicine recognized, liver fibrosis can be reversed, but cirrhosis is irreversible. So, faced with chronic hepatitis, on the one hand, anti-virus is still a fundamental treatment, but on the other hand, on the basis of anti-virus no specific treatment, another important means of treatment must be put on the agenda, namely early antifibrotic diagnosis and treatment. Can block, reduce or reverse liver fibrosis, can greatly improve the prognosis of various liver diseases, early detection of liver fibrosis is the premise of liver fibrosis early intervention. Liver biopsy detection of liver fibrosis "gold standard", its clinical application is still difficult to carry out a wide range of commonly used serum HA, P111P, C1V, LN reaction degree of liver fibrosis, but these indicators do not accurately reflect early liver fibrosis, cause difficulty in clinical diagnosis and treatment of early non-invasive detection of liver fibrosis solved.Recent studies show that, miRNA this post-transcriptional level regulatory factor through the regulation of cell proliferation, apoptosis, lipid metabolism/fatty acid metabolism and a series of related pathways play an important role in the development and progression of many liver diseases. Domestic and foreign scholars in recent years, high-throughput chip, RT-RCR animal liver fibrosis and patients with clinical liver tissue research found that a variety of miRNAs differentially expressed in the process of liver fibrosis. It has been found miR-199a, miR-199a*, miR-200a and miR-200b in CCL4liver fibrosis in animal tissues and human liver fibrosis are differentially expressed, and was significantly associated with liver fibrosis stage. Confirm the differential expression of miRNA in HSC activation and proliferation process21miRNA differentially expressed their activation and proliferation are closely related, for example, over-expression of miR-29and miR-19B can be passed down the expression of pro-fibrotic factor inhibition of the synthesis of ECM. Tip miRNAs may be involved in the regulation of HSC biological behavior and affect the development of liver fibrosis. Visible microRNA who plays an important role in the mechanism of the occurrence and development of chronic liver disease, but can be used for liver fibrosis grading or early diagnosis of blood circulating miRNAs have not been reported.microRNA that information is important to mediate intracellular loop nuclear unusually high stability in the blood, prompted circulation miRNA molecules may become an effective method for non-invasive diagnosis of disease. Some studies reported that miR-122and miR-155may be markers of hepatitis C patients with inflammation-mediated liver injury. Was detected in the plasma samples of patients with chronic hepatitis B the nine miRNAs associated with liver disease, found that the expression level of three (miR-199a, miR-125b and miR-122) was statistically significant, changes in miR-122of the most significant. Tip blood may be present in stable detection of miRNAs has potential applications for early yet to confirm the diagnosis in patients with advanced liver fibrosis.The topics proposed by detecting dimethylnitrosamine (dimethylnitrosamine, DMN)-induced liver fibrosis model rat blood miRNA expression profiling, screening of miRNAs differentially expressed in the process of liver fibrosis in peripheral blood and in the DMN and BDL expression of rat liver fibrosis model validation, bioinformatics and statistical analysis, screening of liver fibrosis specific miRNAs; collection hepatitis B patients with liver fibrosis clinical blood samples to detect the target miRNAs in HBV-infected patients peripheral blood samples to build the ROC curve to calculate the target miRNA diagnostic sensitivity and specificity of liver fibrosis, diagnostic efficiency analysis of the target microRNA in liver fibrosis, by measuring the concentration of peripheral blood microRNA to determine the amount of liver fibrosis, provide a new basis for the early detection of liver fibrosis and methods for qualitative and quantitative monitoring, especially in the early stages of liver fibrosis, which can effectively guide the assessment of anti-fibrosis treatment and efficacy.Parti:the establishment of liver fibrosis model in ratsObjective:Established the DMN and BDL rat liver fibrosis model, grade and stage by the general situation in rats and rat liver pathological histological,and collect different stages of liver fibrosis rats peripheral blood specimens to prepare for the; next step miRNA microarray and PCR validation.Methods:DMN was continuous intraperitoneal injected to SD rats in experimental group, the control group received intraperitoneal injection of the same dose of saline; SD rats were up and down the common bile duct at the hepatic portal ligation (bile duct ligated, BDL), andfrom the middle intermittent, leading to cholestasis, BDL cholestatic liver fibrosis model; regularly observe the general change in rats were sacrificed, and the rats were killed before the pentobarbital anesthetized by intraperitoneal injection, abdominal aortic blood PAXgene TM bloodRNA Tube blood collection tube upside down to mix, allowed to stand at room temperature after4hours,-20℃frozen liver tissue HE staining, Masson and VG special staining and pathological analysis to determine the degree of hepatic fibrosis in rats.Results:(1) With the continuation of the modeling cycle, rat general changes accordingly, the DMN model group:rats in each time period activities and defecation reduce deterioration of body hair density and gloss, body weight and liver weight were lower than the control group; BDL model group:rats activities and defecation reduce hair density and gloss variation, due to cholestasis lead to weight and liver weight increase (2) DMN modeling early due to inflammation in rat liver is slightly larger than the normal group, post-hepatic fibrosis cirrhosis after liver volume is less than the control group, the liver visible on the surface of the gray-yellow nodules; BDL-made modules:rat liver than in normal group increased significantly, hilar a yellow cholestasis, the late liver gradually become tough, no surface miliary nodules (3) HE staining and Masson, VG special staining:control group, hepatic lobule structural integrity of liver cells central venous arranged radially around, no degeneration. The DMN model group were visible liver cells cloudy swelling, degeneration and necrosis, showing that a small amount of inflammatory cell exudation, portal area and lobular proliferation of collagen fibers, and ultimately the formation of fibrous interval and pseudolobule of. The BDL model group the portal area lobular proliferation of collagen fibers, the portal at the bile duct students, eventually forming pseudolobule.(4) Rats peripheral blood collected in PAXgene TM Blood RNA Tube tube, stable traits of gene transcription in vivo due to the blood collection tube containing additives,-20℃can be stored for50months, blood samples instinct saved successfully used for chip testing and the latter part of the verificationConclusion:SD rats DMN intraperitoneal injection can be successfully established DMN liver fibrosis in rats, SD rats up and down the common bile duct at the hepatic portal ligation, resulting in cholestasis can be successfully established BDL cholestatic liver fibrosis model. According to the pathological stage and grade, and rat blood samples are grouped and can be used for chip testing. Part Ⅱ:Liver fibrosis animal model of peripheral blood of specific microRNAs screening and verificationObjective:To study the peripheral blood the circulating miRNA differentially expressed in the process of liver fibrosis, DMN and BDL rat liver fibrosis model further test obvious liver fibrosis miRNAs and as the next step of clinical blood specimens verification target microRNA.Methods:(1) levels of liver fibrosis in rats blood samples selected according to the pre-liver fibrosis pathological staging results, the miRNA microarray preliminary screening of miRNA expression differences in the levels of liver fibrosis in peripheral blood;(2) should be transported in real timethe quantitative PCR target miRNA expression in the peripheral blood of different levels of liver fibrosis in the DMN model group (3) combined reported in the literature, and target gene prediction sites, further selected in high abundance in the peripheral blood circulation, and with the process of liver fibrosissignificantly differentially expressed miRNAs (4) different levels of liver fibrosis in BDL model group, peripheral blood verified.Results:(1) miRNA microarray analysis results show that, with the development of liver fibrosis, liver fibrosis in peripheral blood of37miRNAs differentially expressed the highest level of miR-185upregulation, compared with control group, the expression levels increase the maximum, so choose miR-185in the DMN and BDL rats peripheral blood for further verification (2) real-time quantitative PCR:miR-185in the DMN model of hepatic fibrosis in peripheral blood with liver fibrosis progression and its expression increased significantly; In the early stage of liver fibrosis (F1/F2), advanced fibrosis (F3/F4), respectively,4.30and4.75times higher late than early miR-185expression levels increased, but not statistically significant;(3) Real-time PCR analysis showed that miR-185in the BDL model of liver fibrosis in peripheral blood with liver fibrosis progression and its expression increased significantly; in the early stage of liver fibrosis (F1/F2), advanced liver fibrosis (F3/F4), respectively1.70and1.9times higher late than early miR-185expression levels increased, but not statistically significant.(4) miR-185in DMN and DBL in two models of liver fibrosis, the expression of the same trend, with liver fibrosis was significantly increased, suggesting that miR-185can be used as circulating markers of liver fibrosis.Conclusion:The process of liver fibrosis miRNA differential expression of peripheral blood circulation, miR-185E in DMN and DBL model of hepatic fibrosis in the same trend, with liver fibrosis was significantly increased miR-185can be used as liver fibrosiscycle markers Part III:Circulating miR-185, a important clinical diagnosis role for hepatic fibrosisObjective:Chip screening and verification of two animal models, identified miR-185were detected in the peripheral blood of hepatitis B patients with liver fibrosis, a clear role and value of miR-185in clinical diagnosis.Methods:(1) Statistics collected clinical blood samples) from Fuzhou Military General Hospital to raise the blood samples of patients with hepatitis B liver fibrosis;(2) the use of the expression level of real-time quantitative PCR detection of miR-185in liver fibrosis progression in peripheral blood;patient medical records and the pathology report on the results of the liver biopsy, blood samples are grouped;(4) According to the diagnosis of pathological gold standard, and miR-185expression in clinical blood samples for statistical analysis to build the ROC curve to calculate thediagnosis of different stages of liver fibrosis specificity, sensitivity, area under ROC curve, clear of miR-185in the clinical diagnosis of liver fibrosis and value.Results:(1) In the peripheral blood of patients with HBV infection, miR-185with liver fibrosis progress the expression of a significant increase in FO, F1/F2, F3/F4, respectively1.02,1.65and2.96times higher, but in the FO of (hepatitis, liver fibrosis) did not change significantly, and no statistically significant late than early miR-185expression levels increased, with statistical significance, can be speculated that miR-185may be a quantitative indicator of the accumulation of liver fibrosis (2) The area under the ROC curve of miR-185diagnosis of early liver fibrosis for0.8583,95%CI:0.7274-0.9726, sensitivity75%, specificity95.24%P=.0001276, miR-185can be used as a diagnostic indicator of early liver fibrosis.(3) miR-185diagnosis of advanced fibrosis area under ROC curve of0.9395(95%CI0.8725-1.006), sensitivity87.5%, specificity95.24%, P <0.0001, as a advanced liver fibrosis diagnostic indicator.(4)24cases of HBV in peripheral blood of patients with advanced liver fibrosis detection of the expression of miR-185’s, according to the viral load level of analysis and found that the expression of miR-185in different viral load did not change significantly (P=0.9768), that miR-185in the level of expression and HBV infection and viral load in the peripheral bloodConclusions:Progress of miR-185in the peripheral blood of HBV-infected patients with liver fibrosis in its expression level is significantly increased, and the expression amount of HBV infection and low viral load can be determined by measuring the concentration of peripheral blood miR-185of liver fibrosisof the amount, especially in the early stages of liver fibrosis qualitative and quantitative monitoring, instead of difficult to progress in clinical liver biopsy provides the basis and method for the early detection and treatment of liver fibrosis.