Different Analysis Gene Profile Of Primary Biliary Cirrhosis And Verification On Differential Expression Of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease characterized by the chronic inflammation and destruction of intra-hepatic small bile ducts, leading to liver fibrosis, cirrhosis, and eventual liver failure PBC. Over the past decade, as diagnostic techniques developed and the widespread concern in the PBC, PBC is no longer a rare disease. Its diagnosed rate is rised year by year. Although the pathogenesis of PBC is poorly understood, genetic susceptibility and environment factors in breaking tolerance to autoantigens contribute to the development of PBC .In addition, immunological deregulation has been also involved in the pathogenesis of PBC. Diagnosis of PBC depends on the existence of serum anti-mitochondrial antibody (AMA), ALP abnormity or the typical pathological changes in the liver. These markers are not specific for PBC. This makes early diagnosis, early treatment of PBC encountered a bottleneck.Gene chip technology is developed in recent years, with high sensitivity and high throughput features, it has been used for autoimmune disease in a variety of research. Here we have used Oligonucleotide Array Sequence Analysis for PBC PBMCs mRNA profile, followed by Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) and Flow cytometry analysis (FCM) to verified the differential expression gene by compared with PBC patients and controls, for better diagnosis, therapy and pathogenesis research of PBC. Objective: To explore the differentially mRNA profile expressed in PBC compared with healthy controls, to investigate the new pathogenesis and biomarkers of PBC.Method: Peripheral blood mononuclear cells(PBMCs)were isolated from 3 PBC patients and 3 age and sex matched healthy controls. Total RNA was extracted from PBMCs, then analyzed the differential expressed gene by Oligonucleotide Array Sequence Analysis microarrays(22000 gene).Result: External standard, internal standard and positive control signals were normal, negative control test was negative; housekeeping gene was reproducible, Ratio value of the CV was not more than 0.3; the leakage rate was no more than 3‰, the detection rate was normal. 356 genes differentially expressed in PBC were identified by microarray analysis,in which 296 genes were up-regulated and 60 genes were down-regulated. Signaling pathways involved in cellular immune response, humoral immune response, cell metabolism processes, apoptosis and so on.Conclusion: gene expression patterns in PBC are different from Health controls. Gene microarrays could be of help in investigating the pathogenesis and biomarkers of PBC. Objective: To verificate the natural killer cell mediated cytotoxicity pathway differential expressed gene: FCGR3A,TYROBP and PRF1.Method: We examined the mRNA expression levels of FCGR3A,TYROBP and PRF1 in PBMCs from 80 PBC patients, 55 chronic hepatitis type B (CHB) patients and 68 healthy controls by real-time reverse transcription-polymerase chain reaction analysis (RT-PCR). We further used flow cytometry to study the expression of the FCGR3A and PRF1 protein in the each groups of PBMCs of PBC and controls.Result: The FCGR3A, TYROBP and PRF1 mRNA expression in PBMCs was significantly up-regulated in PBC patients compared with healthy controls. PBC patients had higher expression FCGR3A protein expression on NK cells and PRF1 protein expression on all groups of PBMCs than CHB and healthy controls. The expression of PRF1 protein was related to the serum total bilirubin, GGT and Mayo risk score.Conclusion: The verificated results accord with the Gene chip'. These results indicate that increase expression of genes in the NK cell mediated cytotoxicity pathway in PBC and suggest that this pathway might be involved in the pathogenesis of PBC. PRF1 is a potential biomarker for disease prognosis.