| Inflammatory cell recruitment to the liver is a common feature of live diseases. The key concept is that normal inflammation (for example, inflammation associated with wound healing) is usually self-limiting; however, dysregulation of any of the converging factors can lead to abnormalities and ultimately, pathogenesis this seems to be the case such as immunological liver injury, hepatic fibrosis, cirrhosis and even hepatocellular carcinoma. Recent data have expanded the concept that inflammation response/immune response is a critical component of tumor progression. Many cancers arise from sites of infection, chronic irritation and inflammation. It is now becoming clear that the tumour microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. It seems that immunological liver injury plays an important in affecting the prognosis or progression. However, the mechanisms underlying the pathogenesis of immunological liver injury and following disease progression are poorly defined. Our studies are aiming at obtaining information about important molecules that involved in immunological liver injury, and focusing on the cloning of its specific genes so as to explore its molecular mechanism. Firstly, to insight into the pathogenic molecular pathways in immunological liver injury, we construct the immunological hepatic injury gene library using the suppression-subtractive hybridization (SSH), which is a genome-wide approach that enriches for differentially expressed mRNA transcripts, through comparing gene expression of immunological liver injury tissue and hepatocellular carcinoma tissue. After sequencing and searching for homologies of the subtracted library, numerous genes such as chemokines, apoptosis regulations, cell cycle regulations, anti-oncogen, and stress responses genes were found in this gene repertoire. Up-regulation of immunological liver injury-related genes was identified in the libraries, and some unpublished new genes were found and submitted to the NCBI. This genome-widescanning study discloses the molecular portraits of immunological liver injury and HCC, and provides candidate genes that should help clarify the mechanism of immunological liver injury. Many genes in library such as tumor related genes,cell cycle genes,cell growth genes,transcription factor genes and metabolism genes and so on was found to down-regulate in immunological injured liver tissue.Some genes might be involved in immunological liver injury, but as a chemokine, the CXCL ligand 16 (CXCL 16) might play a role in recruitment of inflammatory cells and result in liver injury. CXCL 16 was a new member of chemokines family, it was indicated to be identical SR-PSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein), which is a transmembrane chemokine, and has four distinct domains: a chemokine domain, mucin-like stalk, transmembrane and cytoplasmic tails, similar to structure of FKN, the first reported transmembrane-type chemokine. The CXCL 16 was recently found to express on APC (antigen presenting cells) and can selectively chemoattract activated CD4+ and CD8+T lymphocytes, which are the CXC chemokine receptor 6 (CXCR6)-expressing lymphocytes. To elucidate the comprehensive chemokines gene expression profiles of these liver tissues, the expression level of other liver-related chemokines were detected in normal, immunological injured liver and HCC liver specimen tissue and compared with the CXCL 16 by real-time quantitative PCR. The results showed that expression of CXCL 16 was significantly up-regulated in the immunological injured liver tissue (p<0.05). Dynamics and closely related changes of CXCL 16 expression are suspected to be involved in the pathogenesis of immunological liver injury.Secondly, to investigate the expression pattern and roles of CXCL 16 in immunological liver injury, the mouse immunological liver injury model was conducted by Bacille Calmette-Guerin (BCG) and Lipopolysaccharide (LPS) induction. The expression pattern and distribution of CXCL 16 was detected in this model by real-time quantitative PCR and immunohistochemistry, further the relationship of expression level of CXCL 16 and extend of liver injury was also evaluated. The results demonstrated that serum ALT level in model quickly increased in BCG primed sequentially LPS challenge at 6h in mice and reached peak at 12h (/7<0.05) . The TNF-a elevated and reached the peak at lh. The pathological examination of liver tissue showed that granunomous was formed after BCG priming and infiltration of amount of inflammatory cells was emerge in BCG primed sequentially LPS challenge at 6h and multiple hepatic necrosis at 24h.These resultsdemonstrated that the mouse immunological liver injury was successfully conducted. Examination of CXCL16 expression showed that CXCL16 was strongly increased in injured liver tissue and distributed in vascular endothelium and hepatic sinusoid endothelium in liver tissue. The expression level of CXCL16 might closely be associated with extent of liver injury.Thirdly, to investigate the pathophysiological roles of CXCL16 and effect of anti-CXCL16 Ab blocking in vivo in immunological liver injury, the prokaryotic expression system was employed to express the murine-derived CXCL16. The CXCL16 cloned from mouse splenocytes was inserted into pET32a, the fusion expression vector. Recombinant pET32-CXCL16 was transformed to E.coli strain BL21. After induction with IPTG, the fusion protein with molecular weight of 29kD was expressed. Soluble supernatant containing fusion protein was affinity-purified with HiTrap Chelating HP Columns. The purified fusion protein maintained the original immunogenicity and biological activity. The antiserum of CXCL16 was generated after immunizing rabbits. The polyclonal antiserum was further purified with affinity chromatography column. Double immunodiffusion assay and neutralization of specific antibody analysis showed that the purified anti-CXCL16 antibody had the characteristic of immune activity and antigen specificity. It could be used directly in immunoblotting and neutralization to the CXCL16 biological activity. Anti-CXCL16 antibody was further administrated in vivo to study whether it would inhibit hepatic necrosis. Administration of anti-CXCL16 Ab before LPS challenge protected the mice from acute liver damage, and accompanied by a significant reduction of both ALT level and number of circulating lymphocytes infiltration, especially CD4+ and CD8+ T cells population. Moreover, both proinflammatory factors such as TNF-a and apoptosis molecule like FasL expression in the liver were decreased after anti-CXCL 16 Ab treatment. These findings suggested that intrahepatic recruitment of CXCR6+ lymphocytes (dominant T cells) to CXCL16 might play an important role in massive hepatic necrosis in immunological liver injury, but the details mechanism need to be further investigated.Finally, CXCL16/SR-PSOX is also a transmembrane chemokine and recruit activated-T cells lymphocytes. Little is known about the role of membrane-anchored CXCL16/SR-PSOX in the immunological liver injury. Due to the immunohistochemical analysis showed that the up-regulated membrane-anchored CXCL16 mostly distributed in sinusoid endothelium and vascular endothelium in the...
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