幽门螺旋杆菌毒力蛋白CagA对B淋巴细胞IgA1分泌和低糖基化的影响

发布时间：2018-05-12 23:21

  本文选题：细胞毒素相关蛋白A + IgA1　； 参考：《泸州医学院》2014年硕士论文

【摘要】：目的：IgA肾病是全世界范围内一种常见的原发性肾小球疾病，在我国已成为导致慢性肾功能衰竭最主要的肾小球疾病，大约25%~50%的患者在疾病诊断后25年内发展到终末期肾衰竭（ESRD）；幽门螺杆菌(Helicobacterpylori，Hp)在中国乃至整个亚太地区人群中具有高感染率，目前已有研究提示Hp感染可能与IgA肾病发病有关，但缺乏相关的实验证据，具体致病机制也不明。本实验通过检测Hp的毒力蛋白——细胞毒素相关基因A蛋白（CagA）对人B细胞分泌的IgA1以及糖基化影响，初步研究Hp与IgA的相关性，并通过检测C1GALT1及Cosmc基因的表达变化，进一步探讨HP参与IgA肾病发生的分子机制，其研究结果可为明确Hp与IgA肾病发生的相关性提供实验依据,并为治疗伴有Hp感染的IgA肾病提供新的思路。方法：以培养的人B淋巴细胞系DAKIKI细胞为研究对象，在剂量依赖实验（给予不同浓度的CagA蛋白刺激，培养48h）和时间依赖实验（以1.6μg/ml的CagA刺激培养24h、48h、72h）中，采用细胞计数法和CCK8法检测细胞增殖情况，，酶联免疫吸附法（ELISA）法检测培养上清中IgA1浓度，HAA凝集素结合试验法检测低糖基化程度，并在最适刺激浓度和作用时间条件下，运用实时荧光定量PCR和Western blot法分别检测细胞内半乳糖基转移酶C1GALT1和伴侣蛋白Cosmc在基因与蛋白水平的表达变化，探讨CagA引起人B淋巴细胞分泌的IgA1异常糖基化的分子机制。结果：剂量依赖试验中，细胞计数与CCK8实验结果显示，与对照组相比，一定浓度范围的CagA可促进B细胞增殖，而较高浓度的CagA（3.2μg/ml）表现为明显的抑制作用，ELISA结果显示，不同浓度的CagA均可促进B细胞分泌IgA1，其中以1.6μg/ml的CagA效果最为显著，HAA凝集素结合试验结果显示，1.6μg/ml和3.2μg/ml的CagA蛋白可明显增加IgA1分子的低糖基化水平；时间依赖试验中，在不同的时间点，1.6μg/ml的CagA蛋白刺激组与对应的对照组相比，均出现IgA1分泌和低糖基化程度的增加，并呈明显的时间依赖性；综合以上结果，最终确定以1.6μg/ml CagA作用48h进行后续机制探讨：实时荧光定量RT-PCR与Westernblot研究结果显示，与正常对照组相比，CagA组与LPS组（12.5μg/ml）的C1GALT1和Cosmc的表达在基因和蛋白水平均出现明显降低（P0.05）。结论：CagA可促进B细胞增殖，引起IgA1分泌增加，并能诱导IgA1分子低糖基化，其机制可能与下调C1GALT1和Cosmc的表达相关，提示幽门螺旋杆菌感染可能在IgA肾病的发病机制中起到重要促进作用，抗幽门螺杆菌或抗CagA治疗可能成为幽门螺杆菌感染的IgA肾病患者新的作用靶点。
[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.

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.31

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1 李甫罡;幽门螺旋杆菌毒力蛋白CagA对B淋巴细胞IgA1分泌和低糖基化的影响[D];泸州医学院;2014年

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