本文關(guān)鍵詞： 原發(fā)性體液免疫缺陷病 常見變異型免疫缺陷病 PLCγ2 免疫球蛋白 SIgA　出處：《大理學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的 原發(fā)性體液免疫缺陷病是原發(fā)性免疫缺陷病(PID)中最常見的一類疾病，與遺傳因素相關(guān)，以反復(fù)感染為主要表現(xiàn)，，其并發(fā)癥嚴(yán)重威脅著患者的健康。近年來(lái)，原發(fā)性體液免疫缺陷病逐漸受到各國(guó)醫(yī)學(xué)界的重視，對(duì)原發(fā)性體液免疫缺陷病的研究也取得了重要的進(jìn)展，但在我們國(guó)家，對(duì)該病的研究遠(yuǎn)遠(yuǎn)落后于發(fā)達(dá)國(guó)家，特別是在云南地區(qū)近乎呈空白狀態(tài)。本研究擬調(diào)查云南地區(qū)耳鼻咽喉感染者中原發(fā)性體液免疫缺陷病的發(fā)病情況，并采用分子生物學(xué)的方法探索B細(xì)胞信號(hào)傳遞分子磷脂酶Cγ2在原發(fā)性體液免疫缺陷患者中的表達(dá)情況，分析原發(fā)性體液免疫缺陷病與PLCγ2的相關(guān)性，為原發(fā)性體液免疫缺陷病的病因診斷及治療提供基礎(chǔ)。 研究方法 1.選取大理學(xué)院附屬醫(yī)院、大理州人民醫(yī)院長(zhǎng)期反復(fù)耳鼻咽喉感染患者作為研究對(duì)象，收集病史及臨床表現(xiàn)，采集患者血清通過(guò)單向免疫擴(kuò)散法分析患者血清免疫球蛋白IgG、IgA、IgM的含量，結(jié)合淋巴細(xì)胞流式細(xì)胞術(shù)的分析結(jié)果對(duì)患者作出診斷。 2.采集患者EDTA-Na2抗凝的外周全血，通過(guò)聚蔗糖/泛影葡胺（淋巴細(xì)胞分離液）密度梯度離心法分離外周血單個(gè)核細(xì)胞（PBMC）。 3采用TRNzol從患者外周血單個(gè)核細(xì)胞中提取總RNA，合成cDNA第一鏈，以β-actin基因表達(dá)作為內(nèi)參照，將cDNA進(jìn)行3倍系列稀釋作為PCR擴(kuò)增的模板，采用半定量RT-PCR分析原發(fā)性體液免疫缺陷病患者PLCγ2在mRNA表達(dá)水平，并與健康人比較，分析其差異。 4采用含有多種蛋白酶抑制劑的細(xì)胞裂解液裂解患者外周血單個(gè)核細(xì)胞，離心棄沉渣取上清制備全細(xì)胞蛋白，BCA法測(cè)定蛋白濃度，細(xì)胞蛋白經(jīng)SDS-PAGE分離后，采用半干電轉(zhuǎn)移至PVDF膜，分別與抗-人PLCγ2、抗-人β-actin抗體反應(yīng)，加ECL顯色劑、曝光，用化學(xué)發(fā)光成像系統(tǒng)直接掃描讀取結(jié)果，分析原發(fā)性體液免疫缺陷病患者PLCγ2與健康人在蛋白水平表達(dá)的差異。 5采集長(zhǎng)期反復(fù)耳鼻咽喉感染患者的唾液,采用抗人SIgA抗體通過(guò)競(jìng)爭(zhēng)ELISA法測(cè)定分泌型IgA（SIgA）在唾液中的含量,結(jié)果采用ELISA Calc軟件進(jìn)行分析計(jì)算,并與健康人比較,分析SIgA在原發(fā)性體液免疫缺陷患者中的含量變化。 研究結(jié)果 1.根據(jù)病史、血清免疫球蛋白測(cè)定結(jié)果及流式細(xì)胞術(shù)分析結(jié)果，在42例長(zhǎng)期反復(fù)耳鼻咽喉感染者中，診斷為普通變異性免疫缺陷病（CVID）患者7例，年齡在4.9歲到40歲之間，其中男性5例，女性2例；7例患者血清免疫球蛋白IgM降低。本次調(diào)查未能發(fā)現(xiàn)其它原發(fā)性體液免疫缺陷病患者。 2.對(duì)來(lái)源于7例CVID患者的PBMC mRNA進(jìn)行半定量RT-PCR檢測(cè)結(jié)果顯示，與健康人比較，PLCγ2基因在轉(zhuǎn)錄水平無(wú)明顯差異。 3.對(duì)上述的7例CVID患者以及其他來(lái)源的4例CVID患者（2例系統(tǒng)性紅斑狼瘡，1例類風(fēng)濕性關(guān)節(jié)炎及1例白細(xì)胞減少癥）通過(guò)western blot檢測(cè)發(fā)現(xiàn)，不同CVID患者的PLCγ2蛋白表達(dá)具有差異性，其中3例患者PLCγ2蛋白表達(dá)水平與正常人比較，表達(dá)有受損，檢出率為3/11（27.27%），其余患者PLCγ2蛋白表達(dá)水平無(wú)明顯受損。 4.長(zhǎng)期反復(fù)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，差異有統(tǒng)計(jì)學(xué)意義；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,差異有統(tǒng)計(jì)學(xué)意義；研究進(jìn)一步發(fā)現(xiàn)，原發(fā)性體液免疫缺陷病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時(shí)，唾液分泌型IgA也降低P=0.0090.05，t=2.816,差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論 在長(zhǎng)期反復(fù)耳鼻咽喉感染患者中發(fā)現(xiàn)7例CVID患者，為首次在云南報(bào)道本病的存在。通過(guò)對(duì)CVID患者PLCγ2的蛋白表達(dá)水平分析顯示，細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)分子PLCγ2在有些CVID發(fā)病中可能具有一定的作用。原發(fā)性體液免疫缺陷病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時(shí)，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.

【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R76

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