本文選題：細(xì)胞毒素相關(guān)蛋白A + IgA1��； 參考：《瀘州醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：IgA腎病是全世界范圍內(nèi)一種常見的原發(fā)性腎小球疾病，在我國已成為導(dǎo)致慢性腎功能衰竭最主要的腎小球疾病，大約25%~50%的患者在疾病診斷后25年內(nèi)發(fā)展到終末期腎衰竭（ESRD）；幽門螺桿菌(Helicobacterpylori，Hp)在中國乃至整個亞太地區(qū)人群中具有高感染率，目前已有研究提示Hp感染可能與IgA腎病發(fā)病有關(guān)，但缺乏相關(guān)的實驗證據(jù)，具體致病機制也不明。本實驗通過檢測Hp的毒力蛋白——細(xì)胞毒素相關(guān)基因A蛋白（CagA）對人B細(xì)胞分泌的IgA1以及糖基化影響，初步研究Hp與IgA的相關(guān)性，并通過檢測C1GALT1及Cosmc基因的表達(dá)變化，進(jìn)一步探討HP參與IgA腎病發(fā)生的分子機制，其研究結(jié)果可為明確Hp與IgA腎病發(fā)生的相關(guān)性提供實驗依據(jù),并為治療伴有Hp感染的IgA腎病提供新的思路。方法：以培養(yǎng)的人B淋巴細(xì)胞系DAKIKI細(xì)胞為研究對象，在劑量依賴實驗（給予不同濃度的CagA蛋白刺激，培養(yǎng)48h）和時間依賴實驗（以1.6μg/ml的CagA刺激培養(yǎng)24h、48h、72h）中，采用細(xì)胞計數(shù)法和CCK8法檢測細(xì)胞增殖情況，，酶聯(lián)免疫吸附法（ELISA）法檢測培養(yǎng)上清中IgA1濃度，HAA凝集素結(jié)合試驗法檢測低糖基化程度，并在最適刺激濃度和作用時間條件下，運用實時熒光定量PCR和Western blot法分別檢測細(xì)胞內(nèi)半乳糖基轉(zhuǎn)移酶C1GALT1和伴侶蛋白Cosmc在基因與蛋白水平的表達(dá)變化，探討CagA引起人B淋巴細(xì)胞分泌的IgA1異常糖基化的分子機制。結(jié)果：劑量依賴試驗中，細(xì)胞計數(shù)與CCK8實驗結(jié)果顯示，與對照組相比，一定濃度范圍的CagA可促進(jìn)B細(xì)胞增殖，而較高濃度的CagA（3.2μg/ml）表現(xiàn)為明顯的抑制作用，ELISA結(jié)果顯示，不同濃度的CagA均可促進(jìn)B細(xì)胞分泌IgA1，其中以1.6μg/ml的CagA效果最為顯著，HAA凝集素結(jié)合試驗結(jié)果顯示，1.6μg/ml和3.2μg/ml的CagA蛋白可明顯增加IgA1分子的低糖基化水平；時間依賴試驗中，在不同的時間點，1.6μg/ml的CagA蛋白刺激組與對應(yīng)的對照組相比，均出現(xiàn)IgA1分泌和低糖基化程度的增加，并呈明顯的時間依賴性；綜合以上結(jié)果，最終確定以1.6μg/ml CagA作用48h進(jìn)行后續(xù)機制探討：實時熒光定量RT-PCR與Westernblot研究結(jié)果顯示，與正常對照組相比，CagA組與LPS組（12.5μg/ml）的C1GALT1和Cosmc的表達(dá)在基因和蛋白水平均出現(xiàn)明顯降低（P0.05）。結(jié)論：CagA可促進(jìn)B細(xì)胞增殖，引起IgA1分泌增加，并能誘導(dǎo)IgA1分子低糖基化，其機制可能與下調(diào)C1GALT1和Cosmc的表達(dá)相關(guān)，提示幽門螺旋桿菌感染可能在IgA腎病的發(fā)病機制中起到重要促進(jìn)作用，抗幽門螺桿菌或抗CagA治療可能成為幽門螺桿菌感染的IgA腎病患者新的作用靶點。
[Abstract]:Objective: IgA nephropathy is a common primary glomerular disease worldwide. In China, it has become the most important glomerular disease in chronic renal failure in our country. About 25%~50% of the patients developed to end-stage renal failure (ESRD) within 25 years of the disease diagnosis, and Helicobacterpylori (Hp) in China and even the whole of subacute renal failure (Helicobacterpylori, Hp) There is a high infection rate among the people in the area, which has been suggested that Hp infection may be related to the pathogenesis of IgA nephropathy, but there is a lack of relevant experimental evidence and the specific pathogenesis is unknown. This experiment was conducted by detecting the effect of the toxic protein of Hp, the cytotoxin related gene A protein (CagA), on the IgA1 and glycosylation of human B cells. To investigate the correlation between Hp and IgA, and to further explore the molecular mechanism of HP participation in the occurrence of IgA nephropathy by detecting the changes in the expression of C1GALT1 and Cosmc genes. The results can provide an experimental basis for identifying the correlation between Hp and IgA nephropathy, and provide new ideas for the treatment of IgA nephropathy associated with Hp infection. Cell line DAKIKI cells were studied in a dose dependent experiment (giving different concentrations of CagA protein stimulation, culture 48h) and time dependent experiments (24h, 48h, 72h) of CagA stimulated by 1.6 micron g/ml, cell counting and CCK8 were used to detect cell proliferation. Enzyme linked immunosorbent assay (ELISA) method was used to detect IgA1 concentration in culture supernatant, HAA coagulating The degree of low glycosylation was detected by the method of combination assay, and the expression of C1GALT1 and chaperone Cosmc in the cells were detected by real-time fluorescence quantitative PCR and Western blot, and the IgA1 differentiation of human B lymphocyte caused by CagA was investigated under the optimum concentration and time of action. Molecular mechanism of normal glycosylation. Results: in the dose dependence test, the cell count and CCK8 test showed that compared with the control group, a certain concentration range of CagA could promote the proliferation of B cells, while the higher concentration of CagA (3.2 mu g/ml) showed significant inhibitory effect. ELISA results showed that CagA in different concentrations could promote the secretion of IgA1 in B cells. The CagA effect of 1.6 mu g/ml was the most significant. The HAA agglutinin binding test showed that the CagA protein of 1.6 g/ml and 3.2 mu g/ml could significantly increase the low glycosylation level of IgA1 molecules. In the time dependent test, the CagA protein stimulation group of 1.6 mu g/ml showed IgA1 secreting and low glycosylation at different time points. The degree was increased, and the time dependence was obvious. In addition to the above results, the follow-up mechanism was determined with 1.6 g/ml CagA action 48h. The results of real-time fluorescence quantitative RT-PCR and Westernblot showed that the expression of C1GALT1 and Cosmc in CagA and LPS group (CagA) was both at the level of gene and protein. There is a significant decrease (P0.05). Conclusion: CagA can promote the proliferation of B cells, increase the secretion of IgA1 and induce the low glycosylation of IgA1 molecules. The mechanism may be related to the expression of C1GALT1 and Cosmc, suggesting that H. pylori infection may play an important role in the pathogenesis of IgA nephropathy, anti Helicobacter pylori or anti CagA treatment. Treatment may become a new target for IgA nephropathy patients with Helicobacter pylori infection.

【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692.31

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