| [Objective]Hepatitis B virus associated glomerulonephritis is the most extrahepatic manifestations of HBV infection. However, the pathogenesis of HBV-GN is still not clear. Most researches show that the pathogenesis of HBV-GN is immune injury.A recent study showed that AIM2is a cytoplasmic double-stranded DNA sensor. Itcould be activated by recognizing cytoplasmic double-stranded DNA and coming into being a immune complex, which triggers IL-1β maturation and secretion through Caspase-1and induces innate immune response. Then AIM2plays an important role in against infection with microbial or viral pathogens in human body. The aims of this study are to explore the role of activation and expression of AIM2in pathogenesis of Hepatitis B virus associated glomerulonephritis.[Methods]1. The human glomerular mesangial (HMC) cell line used in this study was purchased from ScienCell Research Laboratories (California, USA).Cells were cultured and amplified in vitro in tissue culture flask to identify cell phenotype.2. Restriction endonuclease assay Was used to select and identify the positive clones of pcDNA3.0-1.1HBV. Automatic DNA sequencing Was used for the anlysis of HBV sequences. 3.AIM2specific siRNA(1#-3#) were designed and synthesized. The human glomerular mesangial were tmnsfected by using cationic lipid vetors. Determined transfection efficiency by the FAM fluorescent labeling. Cell morphology after transfection was observed by invert microscope. The level of genes expression were valued by RealtimePCR. Screened effective siRNA sequences by Western blot. The SPSS program (version17.0) was used for analysis which segment is the most effective silencer.4. In vitro, AIM2was knocked down with AIM2-siRNA in HBV-infected human glomerular mesangial (HGM) cells. Control cells were infected with either HBV or AIM2-siRNA. Protein and mRNA levels of caspase-1, IL-1β and IL-18in these cell groups were then detected by Western blot and qRT-PCR, respectively. The different expression of AIM2, Caspase-1, IL-1β and IL-18were analyzed by SPSS17.0software.[Results]1. Subculture human glomerular mesangial cells are attach the tissue culture wall well. Under a microscope, HMC cells were homogeneous and transparent, cell body assumes the fibrous, fusiform or elongated, cellular structure is not obvious. It has many radiated during growth, such as flame regularly and conformed to the characteristics of fibroblasts.2. The HBV eukaryotic expression vectors pcDNA3.0-1.1HBVwere verified by endonuclease digestion and DNA sequencing.The report said it has the right sequencing.3. The experiment showed that the transfection efficiency was more than70%. It was observe morphology cells and growth after transfection had no significant effect. The results of Real-time and Western Blot showed both AIM2mRNA and protein level was inhibited most significantly by siRNA-2#.A significant decrease in mRNA levels of gene were observed after24hours, which rebounded after48hours. 4.1n vitro, siRNA-mediated knockdown of AIM2in HBV-infected HGM cells resulted in a2.9-fold decrease in the expression of caspase-1, a3.8-fold decrease of IL-1β and a2.8-fold decrease of IL-18compared to HBV-infected HGM cells without AIM2-siRNA. Similarly, the expressions of caspase-1, IL-1β and IL-18were down-regulated3.4-,4.3-and3.7-fold, respectively, compared to uninfected, AIM2-siRNA-positive HGM cells. Western blot results confirmed a decrease of caspase-1, IL-1β and IL-18protein levels.[Conclusion]AIM2could recognize HBV-DNA and be activated, through Caspase-1activate innate immune system, then release inflammatory factor IL-1β and IL-18,result in Hepatitis B virus associated glomerulonephritis. |
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