AIM2诱导固有免疫在乙肝相关性肾小球肾炎发病机制中的作用

发布时间：2018-05-01 06:11

本文选题：乙肝相关性肾小球肾炎 + 黑素瘤缺乏因子-2　；参考：《山东大学》2014年硕士论文

【摘要】：[背景／目的] 乙型肝炎病毒相关性肾小球肾炎(HBV-GN)是乙型肝炎病毒感染最主要的肝外表现之一。然而HBV-GN的发病机制尚不明确,多数研究认为与免疫损伤有关。黑素瘤缺乏因子2(AIM2是最近研究发现的胞质双链DNA传感器,它能够识别胞质内的双链DNA并被激活,形成免疫复合物,通过Caspase-1途径,促进IL-1p与IL-18前体的成熟及分泌,诱导固有免疫反应,在机体抵御病毒、细菌感染中起重要作用。本研究旨在初步探讨AIM2的激活与表达在IHBV-GN发病机制中的作用。 [方法] 1.于ScienCell Research Laboratories (California,USA)购买人肾小球系膜细胞系(HMC),体外培养,观察细胞形态。 2.将pcDNA3.0-1.1HBV质粒转化大肠杆菌感受态细胞DH5a,经抗性筛选、酶切分析、测序,鉴定质粒。 3.设计合成针对人AIM2基因序列特征设计特异siRNA-(1#—3#),以阳离子脂质体为载体,转染体外培养的人肾小球系膜细胞；FAM荧光标记,测定转染率,观察转染后细胞形态。荧光定量PCR检测siRNA抑制AIM2mRNA水平上表达效果。进一步通过Western blot检测siRNA抑制AIM2蛋白水平表达效果。应用SPSS17.0软件分析AIM2在各组表达的差异,筛选出针对AIM2基因最有效的沉默片段。 4.利用筛选出的针对AIM2基因的有效siRNA序列,干扰人肾小球系膜细胞。将HMC细胞分为AIM2siRNA2#及pcDNA3.0-1.1HBV共转染组,AIM2siRNA control及pcDNA3.0-1.1HBV共转染组,AIM2siRNA2#及pcDNA3.0共转染组,荧光定量PCR与western bolt分别检测各组caspase-1, IL-1β, IL-18的表达。应用SPSS17.0软件分析caspase-1、IL-1β,IL-18在各组表达的差异。评价AIM2基因沉默后对这个炎性网络中其他炎性因子的影响。 [结果] 1.人肾小球系膜细胞传代培养贴壁良好,显微镜下可见,HMC细胞均质、透明,胞体呈纤维状、梭形或长条形,细胞结构不明显,在生长时多呈放射状,火焰状等有规律的走行,符合成纤维细胞特征。 2.pcDNA3.0-1.1HBV质粒转化后随即小量抽提质粒,BamHI酶切,1%琼脂糖凝胶电泳,证实目的基因定向插入成功。对pcDNA3.0-1.1HBV进行测定和同源性分析,证实序列正确。 3.FAM荧光标记后测定转染率在70%以上,观察转染后细胞形态及生长均无明显影响。荧光定量PCR及Western blot结果显示AIM2siRNA2#相对基因抑制水平最强。 4.荧光定量PCR结果显示与HBV组比较,阻断AIM2表达可使caspase-1, IL-1β, IL-18表达量分别下降2.9、3.8、2.8倍。与AIM2siRNA组比较,转染HBV可使caspase-1, IL-1β,IL-18表达量分别下降3.4、4.3、3.7倍,Western blot结果与荧光定量PCR结果一致。 [结论] AIM2通过与HBV-DNA识别并激活,经Caspase-1途径激发固有免疫,释放IL-1β,IL-18等炎性因子,从而导致乙肝病毒相关性肾小球肾炎的肾脏损伤。
[Abstract]:[background / purpose] Hepatitis B virus associated glomerulonephritis (HBV GNN) is one of the most important extrahepatic manifestations of hepatitis B virus infection. However, the pathogenesis of HBV-GN is still unclear, and most studies suggest that it is related to immune injury. Melanoma deficiency factor (2(AIM2) is a recently discovered cytoplasmic double-stranded DNA sensor, which can recognize and activate double-stranded DNA in the cytoplasm, form immune complex, and promote the maturation and secretion of IL-1p and IL-18 precursors through Caspase-1 pathway. Induction of innate immune response plays an important role in resisting viruses and bacterial infections. The purpose of this study was to explore the role of AIM2 activation and expression in the pathogenesis of IHBV-GN. [methods] 1. Human glomerular Mesangial cell line (HMC) was purchased from ScienCell Research Laboratories and cultured in vitro to observe its morphology. 2. The pcDNA3.0-1.1HBV plasmid was transformed into Escherichia coli (E. coli) competent cell line DH 5a. The plasmid was identified by resistance screening, enzyme digestion analysis, sequencing and identification. 3. To design and synthesize a specific siRNA-1 #-3 #-3 #-3 for the sequence characteristics of human AIM2 gene. Using cationic liposome as a vector, the fluorescent labeling of human glomerular Mesangial cells was carried out in vitro. The transfection rate was measured and the morphology of transfected cells was observed. Fluorescence quantitative PCR was used to detect the inhibitory effect of siRNA on the expression of AIM2mRNA. The effect of siRNA on the expression of AIM2 protein was detected by Western blot. The difference of AIM2 expression in each group was analyzed by SPSS17.0 software, and the most effective silencing fragment for AIM2 gene was screened out. 4. The effective siRNA sequence for AIM2 gene was used to interfere with human glomerular Mesangial cells. HMC cells were divided into AIM2siRNA2# and pcDNA3.0-1.1HBV cotransfection group (AIM2 siRNA control) and pcDNA3.0-1.1HBV cotransfection group (AIM2 siRNA2# and pcDNA3.0 cotransfection group). The expression of caspase-1, IL-1 尾 and IL-18 in each group was detected by fluorescence quantitative PCR and western bolt, respectively. SPSS17.0 software was used to analyze the expression of caspase-1, IL-1 尾 and IL-18 in each group. To evaluate the effect of AIM2 gene silencing on other inflammatory factors in this inflammatory network. [results] 1. The cultured human Mesangial cells were well adhered to the wall. Under the microscope, the HMC cells were homogeneous, transparent, fibrous, fusiform or long, and the cell structure was not obvious. In line with the characteristics of fibroblasts. 1% agarose gel electrophoresis was performed immediately after the transformation of 2.pcDNA3.0-1.1HBV plasmids, and a small number of plasmids were extracted from the plasmids BamHI, which confirmed that the target gene was inserted successfully. PcDNA3.0-1.1HBV analysis and homology analysis showed that the sequence was correct. The transfection rate was more than 70% after 3.FAM labeling. The morphology and growth of the transfected cells were not significantly affected. Fluorescence quantitative PCR and Western blot showed that AIM2siRNA2# had the strongest relative gene inhibition. 4. Fluorescence quantitative PCR showed that blocking the expression of AIM2 could decrease the expression of caspase-1, IL-1 尾 and IL-18 by 2.8 times respectively compared with HBV group. Compared with AIM2siRNA group, transfection of HBV decreased the expression of caspase-1 and IL-1 尾 -tir IL-18, respectively. The results of Western blot were consistent with those of fluorescent quantitative PCR. [conclusion] AIM2 is recognized and activated with HBV-DNA, which stimulates innate immunity through Caspase-1 pathway and releases inflammatory factors such as IL-1 尾 -IL-18, which leads to renal injury in hepatitis B virus-associated glomerulonephritis (HBV-associated glomerulonephritis).
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62;R692.31

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