本文選題：乙肝相關(guān)性腎小球腎炎 + 黑素瘤缺乏因子-2　； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：[背景／目的] 乙型肝炎病毒相關(guān)性腎小球腎炎(HBV-GN)是乙型肝炎病毒感染最主要的肝外表現(xiàn)之一。然而HBV-GN的發(fā)病機制尚不明確,多數(shù)研究認為與免疫損傷有關(guān)。 黑素瘤缺乏因子2(AIM2是最近研究發(fā)現(xiàn)的胞質(zhì)雙鏈DNA傳感器,它能夠識別胞質(zhì)內(nèi)的雙鏈DNA并被激活,形成免疫復(fù)合物,通過Caspase-1途徑,促進IL-1p與IL-18前體的成熟及分泌,誘導(dǎo)固有免疫反應(yīng),在機體抵御病毒、細菌感染中起重要作用。本研究旨在初步探討AIM2的激活與表達在IHBV-GN發(fā)病機制中的作用。 [方法] 1.于ScienCell Research Laboratories (California,USA)購買人腎小球系膜細胞系(HMC),體外培養(yǎng),觀察細胞形態(tài)。 2.將pcDNA3.0-1.1HBV質(zhì)粒轉(zhuǎn)化大腸桿菌感受態(tài)細胞DH5a,經(jīng)抗性篩選、酶切分析、測序,鑒定質(zhì)粒。 3.設(shè)計合成針對人AIM2基因序列特征設(shè)計特異siRNA-(1#—3#),以陽離子脂質(zhì)體為載體,轉(zhuǎn)染體外培養(yǎng)的人腎小球系膜細胞；FAM熒光標記,測定轉(zhuǎn)染率,觀察轉(zhuǎn)染后細胞形態(tài)。熒光定量PCR檢測siRNA抑制AIM2mRNA水平上表達效果。進一步通過Western blot檢測siRNA抑制AIM2蛋白水平表達效果。應(yīng)用SPSS17.0軟件分析AIM2在各組表達的差異,篩選出針對AIM2基因最有效的沉默片段。 4.利用篩選出的針對AIM2基因的有效siRNA序列,干擾人腎小球系膜細胞。將HMC細胞分為AIM2siRNA2#及pcDNA3.0-1.1HBV共轉(zhuǎn)染組,AIM2siRNA control及pcDNA3.0-1.1HBV共轉(zhuǎn)染組,AIM2siRNA2#及pcDNA3.0共轉(zhuǎn)染組,熒光定量PCR與western bolt分別檢測各組caspase-1, IL-1β, IL-18的表達。應(yīng)用SPSS17.0軟件分析caspase-1、IL-1β,IL-18在各組表達的差異。評價AIM2基因沉默后對這個炎性網(wǎng)絡(luò)中其他炎性因子的影響。 [結(jié)果] 1.人腎小球系膜細胞傳代培養(yǎng)貼壁良好,顯微鏡下可見,HMC細胞均質(zhì)、透明,胞體呈纖維狀、梭形或長條形,細胞結(jié)構(gòu)不明顯,在生長時多呈放射狀,火焰狀等有規(guī)律的走行,符合成纖維細胞特征。 2.pcDNA3.0-1.1HBV質(zhì)粒轉(zhuǎn)化后隨即小量抽提質(zhì)粒,BamHI酶切,1%瓊脂糖凝膠電泳,證實目的基因定向插入成功。對pcDNA3.0-1.1HBV進行測定和同源性分析,證實序列正確。 3.FAM熒光標記后測定轉(zhuǎn)染率在70%以上,觀察轉(zhuǎn)染后細胞形態(tài)及生長均無明顯影響。熒光定量PCR及Western blot結(jié)果顯示AIM2siRNA2#相對基因抑制水平最強。 4.熒光定量PCR結(jié)果顯示與HBV組比較,阻斷AIM2表達可使caspase-1, IL-1β, IL-18表達量分別下降2.9、3.8、2.8倍。與AIM2siRNA組比較,轉(zhuǎn)染HBV可使caspase-1, IL-1β,IL-18表達量分別下降3.4、4.3、3.7倍,Western blot結(jié)果與熒光定量PCR結(jié)果一致。 [結(jié)論] AIM2通過與HBV-DNA識別并激活,經(jīng)Caspase-1途徑激發(fā)固有免疫,釋放IL-1β,IL-18等炎性因子,從而導(dǎo)致乙肝病毒相關(guān)性腎小球腎炎的腎臟損傷。
[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).
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.62;R692.31

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