耳鼻咽喉感染者中原发性体液免疫缺陷病的调查及与PLCγ2的相关性研究

发布时间：2018-01-20 23:45

本文关键词： 原发性体液免疫缺陷病常见变异型免疫缺陷病 PLCγ2 免疫球蛋白 SIgA　出处：《大理学院》2012年硕士论文　论文类型：学位论文

【摘要】：研究目的原发性体液免疫缺陷病是原发性免疫缺陷病(PID)中最常见的一类疾病，与遗传因素相关，以反复感染为主要表现，，其并发症严重威胁着患者的健康。近年来，原发性体液免疫缺陷病逐渐受到各国医学界的重视，对原发性体液免疫缺陷病的研究也取得了重要的进展，但在我们国家，对该病的研究远远落后于发达国家，特别是在云南地区近乎呈空白状态。本研究拟调查云南地区耳鼻咽喉感染者中原发性体液免疫缺陷病的发病情况，并采用分子生物学的方法探索B细胞信号传递分子磷脂酶Cγ2在原发性体液免疫缺陷患者中的表达情况，分析原发性体液免疫缺陷病与PLCγ2的相关性，为原发性体液免疫缺陷病的病因诊断及治疗提供基础。研究方法 1.选取大理学院附属医院、大理州人民医院长期反复耳鼻咽喉感染患者作为研究对象，收集病史及临床表现，采集患者血清通过单向免疫扩散法分析患者血清免疫球蛋白IgG、IgA、IgM的含量，结合淋巴细胞流式细胞术的分析结果对患者作出诊断。 2.采集患者EDTA-Na2抗凝的外周全血，通过聚蔗糖/泛影葡胺（淋巴细胞分离液）密度梯度离心法分离外周血单个核细胞（PBMC）。 3采用TRNzol从患者外周血单个核细胞中提取总RNA，合成cDNA第一链，以β-actin基因表达作为内参照，将cDNA进行3倍系列稀释作为PCR扩增的模板，采用半定量RT-PCR分析原发性体液免疫缺陷病患者PLCγ2在mRNA表达水平，并与健康人比较，分析其差异。 4采用含有多种蛋白酶抑制剂的细胞裂解液裂解患者外周血单个核细胞，离心弃沉渣取上清制备全细胞蛋白，BCA法测定蛋白浓度，细胞蛋白经SDS-PAGE分离后，采用半干电转移至PVDF膜，分别与抗-人PLCγ2、抗-人β-actin抗体反应，加ECL显色剂、曝光，用化学发光成像系统直接扫描读取结果，分析原发性体液免疫缺陷病患者PLCγ2与健康人在蛋白水平表达的差异。 5采集长期反复耳鼻咽喉感染患者的唾液,采用抗人SIgA抗体通过竞争ELISA法测定分泌型IgA（SIgA）在唾液中的含量,结果采用ELISA Calc软件进行分析计算,并与健康人比较,分析SIgA在原发性体液免疫缺陷患者中的含量变化。研究结果 1.根据病史、血清免疫球蛋白测定结果及流式细胞术分析结果，在42例长期反复耳鼻咽喉感染者中，诊断为普通变异性免疫缺陷病（CVID）患者7例，年龄在4.9岁到40岁之间，其中男性5例，女性2例；7例患者血清免疫球蛋白IgM降低。本次调查未能发现其它原发性体液免疫缺陷病患者。 2.对来源于7例CVID患者的PBMC mRNA进行半定量RT-PCR检测结果显示，与健康人比较，PLCγ2基因在转录水平无明显差异。 3.对上述的7例CVID患者以及其他来源的4例CVID患者（2例系统性红斑狼疮，1例类风湿性关节炎及1例白细胞减少症）通过western blot检测发现，不同CVID患者的PLCγ2蛋白表达具有差异性，其中3例患者PLCγ2蛋白表达水平与正常人比较，表达有受损，检出率为3/11（27.27%），其余患者PLCγ2蛋白表达水平无明显受损。 4.长期反复耳鼻咽喉感染者的患者唾液SIgA水平低于健康者t=-2.818，P=0.0090.05，差异有统计学意义；耳鼻咽喉感染者中血清免疫球蛋白降低者唾液SIgA水平低于健康人群P=0.0020.05，t=3.385,差异有统计学意义；研究进一步发现，原发性体液免疫缺陷病CVID患者唾液的SIgA低于健康人群，即CVID患者血清免疫球蛋白降低的同时，唾液分泌型IgA也降低P=0.0090.05，t=2.816,差异有统计学意义。结论在长期反复耳鼻咽喉感染患者中发现7例CVID患者，为首次在云南报道本病的存在。通过对CVID患者PLCγ2的蛋白表达水平分析显示，细胞信号转导分子PLCγ2在有些CVID发病中可能具有一定的作用。原发性体液免疫缺陷病CVID患者唾液的SIgA低于健康人群，即CVID患者血清免疫球蛋白降低的同时，唾液分泌型IgA也降低。
[Abstract]:research objective
Primary humoral immunodeficiency disease is primary immunodeficiency disease (PID) is a kind of disease in the most common, associated with genetic factors, with repeated infection were the main manifestations of the complications are a serious threat to the health of patients. In recent years, the primary humoral immunodeficiency disease gradually by the medical community's attention on study primary humoral immunodeficiency disease has also made significant progress, but in our country, to study the disease is far behind the developed countries, especially in the area of Yunnan is nearly blank. This study intends to investigate the incidence cases of primary immunodeficiency disease in Yunnan area of Otorhinolaryngology infection, and using method molecular biology to explore B cell signaling molecules phospholipase C gamma 2 in patients with primary humoral immunodeficiency in the expression and correlation analysis of primary humoral immunodeficiency disease and PLC gamma 2, primary body The basis of the etiological diagnosis and treatment of liquid immunodeficiency disease (immunodeficiency disease) is provided.
research method
The 1. Affiliated Hospital of Dali University, Dali People's Hospital otolaryngology repeated infection patients as the research object, medical history and clinical manifestations, serum were collected for analysis of serum immunoglobulin in patients with IgG by immunodiffusion method IgA, the content of IgM, combined with lymphocytes by flow cytometry analysis results of patients with a diagnosis.
2. peripheral blood was collected from patients with EDTA-Na2 anticoagulation, and peripheral blood mononuclear cells (PBMC) were separated by polysucrose / meglumine dihydrochloride (lymphocyte separation liquid) density gradient centrifugation.
3 using TRNzol to extract total RNA from peripheral blood mononuclear cells of the patients, the first strand cDNA was synthesized by beta, -actin gene expression as a reference, the cDNA series of 3 times dilution as a template for PCR amplification, using semi quantitative RT-PCR analysis of primary immunodeficiency disease in 2 patients PLC gamma mRNA expression level, and compared with healthy people, analysis of their differences.
4 the blood mononuclear cells, cell lysis with protease inhibitors containing a variety of peripheral, centrifuged sediment supernatant preparation of whole cell protein, protein concentration was determined by BCA method, cell protein by SDS-PAGE after separation by semi dry electro transferred to PVDF membrane, respectively with anti human PLC anti human gamma 2. Beta -actin antibody response, ECL chromogenic agent, exposure, direct scanning results using chemiluminescence imaging system, analysis of the differences of primary humoral immunodeficiency disease patients and 2 healthy people PLC gamma expression at the protein level.
The 5 acquisition of long-term repeated ent infection in patients with salivary secretory IgA, determined by competitive ELISA method using anti human SIgA antibody (SIgA) content in saliva, using the ELISA Calc software for analysis and calculation, compared with healthy people, analysis of SIgA in primary humoral immunity changes in patients with disease of defect.
Research results
1. according to the history, and the analysis result of flow cytometry detection results of serum immunoglobulin, in 42 cases of recurrent ENT infection in the diagnosis of common variable immunodeficiency disease (CVID) patients with 7 cases, aged between 4.9 to 40 years old, there were 5 males and 2 females; 7 cases the serum immunoglobulin IgM decreased. This investigation failed to find other patients with primary immunodeficiency disease.
2. PBMC mRNA from 7 cases of CVID patients were detected by semi quantitative RT-PCR test. Compared with healthy people, there was no significant difference in the transcriptional level of PLC gamma 2 gene.
4 cases of 3. CVID patients in 7 patients with CVID and the other sources (2 cases of systemic lupus erythematosus, 1 patients with rheumatoid arthritis and 1 cases of leukopenia) by Western blot assay showed that PLC gamma CVID protein expression in 2 patients with different has the difference, of which 3 cases of patients with PLC gamma 2 protein expression levels compared with healthy controls, the expression is damaged, the detection rate was 3/11 (27.27%), the remaining patients PLC gamma 2 protein expression level was not injured.
4. recurrent ear nose throat infection in patients with salivary SIgA level is lower than the healthy subjects t=-2.818, P=0.0090.05, the difference was statistically significant; ear nose throat infection serum immunoglobulin in saliva decreased SIgA levels lower than the healthy population of P=0.0020.05, t=3.385, the difference was statistically significant; further study found the primary humoral immunodeficiency disease CVID the saliva of patients with SIgA is lower than that of healthy people, patients with CVID serum immune globulin decreased at the same time, salivary secretory IgA also decreased P=0.0090.05, t=2.816, the difference was statistically significant.
conclusion
In the long-term repeated ent infection were found in 7 patients with CVID, which was first reported in Yunnan in the presence of the disease. Patients with CVID PLC gamma 2 protein expression analysis showed that the cell signal transduction molecule PLC gamma 2 in some of the pathogenesis of CVID may play a role. The primary humoral immunodeficiency disease CVID the saliva of patients with SIgA is lower than that of healthy people, patients with CVID serum immune globulin decreased at the same time, salivary secretory IgA also decreased.

【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R76

【参考文献】