| Objective It was known that NK cells played an important role in liver damage caused by hepatitis B virus(HBV). Some cytokine and receptor genes expressed abnormally on immunocytes may increase cytotoxicity of NK cells on cells with viral infections and may be associated with the pathogenesis of fulminant hepatitis B(FHB). In recent years the effect of gene expression in host cells have been thought playing a major role in the development and result of inflamation and immune response,and attract more and more attention.So far,little is known about the relation between genes expression on immunocytes and the development of fulminant hepatic failure.In this study,we investigated the relationship between the gene expression on immunocytes (e.g.NK cells)and the development of severe hepatic damage through the screening of genes differential expression on PBMC in patients with FHB. The aim of our experiment was to study the immunopathogenesis of FHB by investigating gene expression and regulation of cell receptor on immunocytes,that will open a new idea and bright prospects in the prevention and treatment of FFTB.Methods At first,high-density microarrays consisting of 8192 human cDNAs were applied to analyze gene expression using labeled cDNAs hybridization of PBMC which prepared from 10 patients with fulminant hepatitis B(FHF) and 10 patients with asymtomatic HBsAgcarries(ASC).Relative expression ratios of each genes were obtained by comparing hybridization of Cy5-labeled cDNA from FHF and Cy3-labeled cDNA from ASC and scanning on a GenePix 4000B,and then by quantitative analysis of genes differential expression using ImaGeneS.O software.The differential expression of target gene were determined in the PBMCs and liver issues obtained from FHB > CHB >, ASC patients and normal peoples by RT-PCR and histochemistry S-P method.Meanwhile, NK cells were sorted after those were cultured and stimulated by PHA and IL-2,and differential expression of target gene were quantitatively determined in patients with FHB> ASC and normal peoples as control by Flow cytometry and immunofluorescent techniques. NK cytolysis on target cells of HepG2, K562 and CNE cells and after NK cells were blocked with specific antibody were analyzed by MTT colorimetry.Finally,an eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique,and was confirmed by double restriction enzymes digestion > PCR and DNA sequence analysis. Then the recombinant plasmid with lipofectamine encapsuled was transfected into CHO cells, and screening by G418 selective culture medium. Finally the CHO cells transfected with recombinant eukaryotic expression plasmid stably were obtained,and the transfected CHO cells containing expressive target gene were confirmed by RT-PCR and Western Blotting techniques.Then the effect of eukaryotic expressionplasmid containing target gene inducing NK cytolysis was analyzed by MTT colorimetry. Results1. Results of high-densty microarrays analysis Out of 8192 genes,249(3%) cellular genes in the FHB were identified as differentially expressed that 2-fold more than in the ASC.lt included 81 up-regulated genes and 168 down-regulated genes.Among 249 genes,total 79% have been found related with the cell signaling and transduction,cell cycle and metabolism, apoptosis and inflammatory response and immunity-related genes.2. Expression of NKG2D mRNA In PBMC the level of expression of NKG2D mRNA among the four groups was gradually down from the group of FHB > then the groups of CHEk normal control to ASC in order of level.In group FHB the level of expression of NKG2D mRNA in PBMC was significantly higher than the other three groups(P<0.01).3. Histochemistry analysis The level of expression of NKG2D gene on immunocytes of hepatic portal areas was significantly higher in the group FHB than in the group CHB and normal control (P<0.01),and in the group CHB(G3/G4) the level of expression of NKG2D was higher than in the group control(P<0.01).But,there...
|
PDF Full Text Request