| Primary biliary cirrhosis (PBC) is characterized by an immune mediated, irreversible destruction of the small intrahepatic bile ducts leading to progressive liver cirrhosis and frequently to liver failure. In past, PBC has not been paid enough attention in our country, but the incidence of detected cases of PBC has been increasing in recent years. So it is necessary to reinforce the study of pathogenesis of PBC for developing new therapeutic measures.The course of PBC is variable and an early diagnosis is desirable to identify individuals with rapidly progressing disease, to initiate adequate therapeutic measures and to evaluate the necessity of liver transplantation. Serological tests represent the single most important diagnostic feature of PBC because liver histology, biochemistry, or clinical syndrome alone are not reliable in this respect. AMA (anti-M2) are a serologic hallmark of PBC. AMA in PBC are directed to the three ketoacid dehydrogenase complexes, and predominantly to E2 subunit of pyruvate dehydrogenase complex (PDC-E2) and E3-binding protein (E3BP) of PDC. Autoantibodis to PDC-E2 and E3BP can be detected in sera of 90-95% of PBC patients. In order to develop the more specific, effcient and economical diagnostic approach and provide basis for study of pathogenesis of PBC, we clonedand expressed PDC-E2 and PDC- E3BP in E.coli. According to the published sequence, the PDC-E2 cDNA without precursor sequence was amplified with RT-PCR method as two segments (1.3kb and 0.6kb), which were linked together and cloned into a prokaryotic expression plasmid -pExSec I to construct recombinant plasmid pExSecI/ PDC-E2. We also cloned PDC-E3BP cDNA (1.35kb) into pET28a(+) to construct recombinant prokaryotic expression plasmid pET28a(+)/E3BP. The recombinant protein of PDC-E2 and PDC-E3BP were high effectively expressed in E.coli BL21 ( DE3 ) plysE with IPTG induction under different condition. The expressed protein existed dissolvablely. By using Western Blot analyses, the expressed products could be identified by the specific self-antibodies in the sera of PBC patients. The ELISA plate were coated with expressed protein to screen sera of 2900 patients with various liver diseases including PBC, and 800 healthy blood donors. The result showed that the positive rate of autoantibodies against these two recombinant protein in sera of PBC patients is 93.3%, which was much higher than that of patients with other liver diseases and healthy people as low positive rate or zero. The ELISA results indicated the two expressed protein could highly specifically bind the autoantibodies in the sera of PBC patients. Compared to the anti-M2 ELISA kit of Euroimmune company, the expressed proteins were the similar on the effectiveness of helping diagnosis of PBC, while the cost of expressed pretein is much lower than the former, which make this two expressed protein having good prospect in developing kit for detecting PBC-specific AMA.The accumulating data suggested that the autoimmune attack on the small intrahepatic bile ducts of PBC patients is mediated by cellular mechanisms and PDC-E2 was supposed to be one of the most possible target antigen that initiate this process. In this research, we evaluated the peripheral blood T cell responses to PDC-E2 and its possible role in pathogenesis of PBC by using PDC-E2 to stimulate peripheral bloodmononuclear cells (PBMC). In our results, the proliferation of PBMC by PDC-E2 was oberserved in 10 out of 12 AMA or anti-M2 positive PBC patients, and most of them (10/12) secreted higher level cytokine of Thl subset (IL-2, IFN-y) and TNF-a, parti cully in four PBC patients who were in early stage. The increase of cytokine of Th2 subset (IL-4, IL-10 ) and TNF-a were only seen in two PBC patients who are in late or middle stage. These results not only suggests possibility of PDC-E2 as a target antigen to induce autoimmune demage in PBC, but also suggested existence of imbalance of Thl/ Th2 subset and that imbalance of Thl/ Th2 subset changes during the course of...
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