本文選題：慢性肉芽腫病 + 臨床特征��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：第一部分慢性肉芽腫病患兒臨床與分子特征研究目的:探討26例慢性肉芽腫病(CGD)患兒的臨床特征,實驗室檢查結果及基因突變特征。方法:1.收集2015年6月-2017年2月在重慶醫(yī)科大學附屬兒童醫(yī)院收治的26例CGD患兒臨床資料和實驗室檢查結果并分析總結。2.流式細胞術分析患兒中性粒細胞呼吸爆發(fā)功能,并計算中性粒細胞氧化指數NOI(刺激后平均熒光強度/刺激前平均熒光強度)。3.PCR直接測序法分析患兒CGD相關基因(CYBB、CYBA、NCF1、NCF2)。結果:1.一般情況:26例患兒均為男性,15例(57.7%)來自我國西南地區(qū),平均起病年齡2.3月,平均診斷年齡1.4歲,14例(53.8%)有家族史(男性親屬早年夭折)。2.臨床表現:26例患兒均有反復發(fā)熱,肺炎(24例,92.3%)、腹瀉(13例,50%)、皮膚黏膜及皮下組織感染(14例,53.8%)、肝脾腫大、肝功能異常(12例,46.2%)為本組患兒最常見的臨床表現。21例接種卡介苗的患兒中14例(66.7%)發(fā)生BCG接種異常反應(卡疤化膿、BCG淋巴結炎、肺結核)。此外,3例(11.5%)發(fā)生肝膿腫,2例(7.7%)常患尿路感染3.實驗室檢查:本組患兒多出現外周血白細胞總數增高,以中性粒細胞為主,多有CRP增高。淋巴細胞分類未見明顯異常,免疫球蛋白正�；虼鷥斝栽龈�,其中9例(34.6%)出現Ig E明顯增高。除2例患兒NBT正常外,其余21例(80.8%)NBT均明顯降低,多數PMA刺激后為0。僅50%患兒找到致病病原菌。4.呼吸爆發(fā)試驗及基因突變分析:患兒NOI多數低于2(1.365±0.1018),明顯低于正常值(正常人100),患兒母親NOI正常或低于正常值;21例(80.8%)患兒發(fā)現CYBB基因突變,1例(3.8%)CYBA突變,4例(15.4%)尚未明確致病基因,發(fā)現15名XLR-CGD攜帶者。結論:CGD患兒多于生后即發(fā)生嚴重的細菌及真菌感染,患兒伴有BCG接種異常反應及男性親屬早年夭折家族史,白細胞總數顯著增高,以中性粒細胞為主,NBT異常,免疫球蛋白正常或升高,則需警惕CGD。流式細胞術檢測中性粒細胞呼吸爆發(fā)功能可快速診斷CGD,基因檢測可進一步確診患者、發(fā)現攜帶者,有產前診斷意義。第二部分慢性肉芽腫病患兒記憶B細胞減少的相關因素研究目的:探討CGD患兒是否存在外周血記憶B細胞(MBC)減少的現象,尋找其減少的原因;并探索IFN-γ治療CGD患兒,減少反復感染的可能機制,為臨床運用IFN-γ治療和預防反復感染提供理論依據。方法:1.流式細胞術檢測CGD患兒MBC百分比、CD4+T細胞表面CD40L表達量、Tfh細胞及其表面PD-1百分比。2.不同濃度s CD40L(1μg/ml、1.5μg/ml、2μg/ml)與正常兒童外周血PBMC共同刺激培養(yǎng)24h、36h、48h,流式細胞術檢測MBC數量的變化。3.分別用不同濃度的外源性H2O2(10μmol/L、30μmol/L)、IFN-γ(2000U/ml、3000U/ml)與CGD患兒外周血PBMC共同刺激培養(yǎng)24h,流式細胞術檢測MBC數量的變化。結果:1.CGD患兒MBC百分比(1.267%±0.1416%)較健康對照兒童(2.824%±0.27%)明顯降低(P0.0001)。2.CGD患兒CD4+T細胞表面CD40L百分比(8.186%±2.736%)較健康對照兒童(46.43%±4.619%)明顯降低(P0.0001)。3.CGD患兒外周血Tfh細胞(5.625%±0.5005%)與健康兒童(6.269%±1.177%)無顯著差異(P=0.5614)。PD-1百分比(9.039%±1.984%)與健康兒童(18.26%±10.11%)無顯著差異(P=0.7546)。4.外源性補充s CD40L不能促進健康兒童MBC增高,MBC百分比隨培養(yǎng)時間延長逐漸降低(P=0.0028)。5.外源性補充H2O2、IFN-γ對CGD患兒MBC無明顯刺激作用,相反,MBC百分比隨培養(yǎng)時間延長逐漸降低(P0.0001)。結論:CGD患兒外周血MBC減少可能是患兒反復感染的原因,MBC減少可能與CD4+T細胞表面CD40L表達減少相關,雖然CGD患兒外周血Tfh細胞數量正常,但仍需進一步分析Tfh細胞表面CD40L表達情況以明確Tfh細胞功能是否正常。IFN-γ并不通過刺激CGD患兒外周血MBC增殖發(fā)揮治療作用。第三部分3例白細胞黏附分子缺陷病Ι型的臨床及分子特征分析目的:探討3例白細胞黏附分子缺陷病Ι型(LAD-1)患兒的臨床特征和CD18蛋白表達異常及基因突變特征。方法:1.總結3例患兒臨床資料,常規(guī)免疫學篩查除外常見原發(fā)性免疫缺陷病。2.流式細胞術檢測白細胞表面CD18分子。3.PCR測序分析患兒及其父母ITGB2基因。結果:1.臨床表現:患兒均自新生兒起以臍炎起病,之后經歷反復皮膚黏膜及軟組織感染(肺炎、中耳炎、鵝口瘡、牙齦炎伴乳牙早脫),其中2例(例1、例2)有臍帶脫落延遲史(21天),2例(例1、例3)有慢性皮膚感染及傷口愈合延遲(1月),1例(例1)家族中有早年夭折患兒。2.實驗室檢查:患兒均有外周血白細胞總數顯著增高,以中性粒細胞為主,因反復感染,常伴有貧血,免疫球蛋白Ig G、Ig A、Ig M增高,淋巴細胞分類、NBT無明顯異常。3.CD18流式分析:患兒白細胞表面CD18分子表達明顯降低,例2為重度缺陷(CD18在淋巴細胞、中性粒細胞、單核細胞上分別為0、0.23%、0),例1、例3為中度缺陷(例1:9.49%、0.04%、0.45%;例3:10.14%、0.67%、2.54%),但所有父母CD18表達均90%。4.ITGB2基因分析:發(fā)現5種突變位點(c.167_168ins GG、c.1884CA、c.533CT、c.817GC、c.1768TC),其中2種為新型突變(例1 c.167-168ins GG、c.1884CA),并發(fā)現5名攜帶者。結論:自幼反復嚴重皮膚黏膜及軟組織感染,尤其伴有臍炎、臍帶脫落延遲,傷口愈合延遲,反復牙齦炎伴乳牙早脫,白細胞總數顯著增高,以中性粒細胞為主,伴免疫球蛋白正常或升高的患兒需警惕LAD-1。流式細胞術檢測白細胞表面CD18分子可快速診斷LAD-1,ITGB2基因分析是診斷的金標準。
[Abstract]:Part 1 clinical and molecular characteristics of chronic granulomatosis children: To investigate the clinical features, laboratory results and gene mutation characteristics of 26 cases of chronic granulomatosis (CGD). Methods: 1. the clinical data and laboratory examination of 26 children with CGD in children's Hospital Affiliated to Medical University Of Chongqing in February June 2015 were collected. Results and analysis and summary of.2. flow cytometry analysis of children's neutrophils respiratory burst function, and calculate neutrophils oxidation index NOI (average fluorescence intensity after stimulation / mean fluorescence intensity before stimulation).3.PCR direct sequencing analysis of children CGD related genes (CYBB, CYBA, NCF1, NCF2). Results: 1. general cases: 26 cases of children are all male, 15 Cases (57.7%) were from the southwest of China. The average age of onset was 2.3 months, the average age of diagnosis was 1.4 years, and 14 cases (53.8%) had a family history of.2.. 26 cases had recurrent fever, pneumonia (24 cases, 92.3%), diarrhea (13 cases, 50%), skin mucosa and subcutaneous tissue infection (14, 53.8%), liver splenomegaly, hepatic dysfunction (12) For example, 46.2%) the most common clinical manifestations of children in this group were 14 cases (66.7%) of.21 vaccinated with BCG (66.7%) with abnormal BCG reaction (card scars purulent, BCG lymphadenitis, pulmonary tuberculosis). In addition, 3 cases (11.5%) had liver abscess, 2 cases (7.7%) often suffered from urinary tract infection 3. laboratory examination: this group had more peripheral blood leukocyte count. Neutrophils were dominant and CRP increased. There was no obvious abnormal lymphocyte classification and normal or compensatory immunoglobulin, of which 9 cases (34.6%) showed significant increase in Ig E. Except 2 cases of NBT normal, 21 cases (80.8%) of NBT were significantly decreased, and 0. of only 50% children after PMA stimulation found pathogenic bacteria.4. respiratory outbreak test and Gene mutation analysis: the majority of NOI in children was less than 2 (1.365 + 0.1018), obviously lower than normal value (normal person 100), children's mother NOI was normal or lower than normal value; 21 cases (80.8%) found CYBB gene mutation, 1 cases (3.8%) CYBA mutation, 4 cases (15.4%) have not clearly caused the disease gene, and found 15 XLR-CGD carriers. Conclusion: CGD children are more severe than after birth. Severe bacterial and fungal infection, children with abnormal reaction of BCG inoculation and the history of premature death of male relatives, the total number of leukocytes increased significantly, with neutrophils dominated, NBT abnormal, and immunoglobulin normal or elevated, CGD. flow cytometry should be vigilant for detecting neutrophils respiratory burst function for rapid diagnosis of CGD, gene detection can be entered. Second cases of chronic granulomatosis children with chronic granulomatosis related factors of memory B cell reduction: To investigate whether there is a decrease in peripheral blood memory B cells (MBC) in children with CGD, and to explore the possible mechanism of IFN- gamma in the treatment of children with CGD and to reduce the possible mechanism of repeated infection. To provide a theoretical basis for the clinical use of IFN- gamma therapy and prevention of recurrent infection. Methods: 1. flow cytometry was used to detect the percentage of MBC in children with CGD, the CD40L expression on the surface of CD4+T cells, Tfh cells and the PD-1 percentage.2. on the surface of the Tfh cells and s CD40L (1 mu g/ml, 1.5 mu g/ml, 2 mu) together with normal children's peripheral blood. The changes in the number of MBC were detected by cytometer..3. was used in different concentrations of exogenous H2O2 (10 mol/L, 30 mu mol/L), IFN- gamma (2000U/ml, 3000U/ml) and CGD children's peripheral PBMC co stimulatory culture 24h. Flow cytometry was used to detect the number of MBC. The results showed that the percentage of children (1.267% + 0.1416%) was significantly lower than that of healthy controls (2.824% + 0.27%). The percentage of CD40L on the surface of CD4+T cells in children with low (P0.0001).2.CGD (8.186% + 2.736%) was significantly lower than that in healthy control children (46.43% + 4.619%). There was no significant difference between the peripheral blood Tfh cells (5.625% + 0.5005%) and healthy children (6.269% + 1.177%) in children (P0.0001).3.CGD (P= 0.5614).PD-1 percentage (9.039% + 1.984%) and healthy children (18.26% + 10.11%). The difference (P=0.7546).4. exogenous s CD40L could not promote the increase of MBC in healthy children, the percentage of MBC was gradually reduced with the prolongation of culture time (P=0.0028).5. exogenous H2O2, IFN- gamma had no obvious stimulation effect on CGD child MBC, but on the contrary, the percentage decreased gradually with the prolongation of culture time. Conclusion: the decrease of peripheral blood in children is reduced. It can be the cause of recurrent infection in children. MBC reduction may be associated with the decrease of CD40L expression on the surface of CD4+T cells. Although the number of Tfh cells in peripheral blood of children with CGD is normal, it is still necessary to further analyze the CD40L expression on the surface of Tfh cells to determine whether Tfh cell function is normal.IFN- gamma and does not pass the peripheral blood MBC proliferation of children with CGD. Clinical and molecular characteristics of 3 cases of leukocyte adhesion molecular defect disease (LAD-1). Objective: To investigate the clinical features, the abnormal expression of CD18 protein and the characteristics of gene mutation in 3 cases of leukocyte adhesion molecular defect disease (LAD-1). Methods: 1. to sum up the clinical materials of 3 cases of children, except for common primary immunology except routine immunological screening. Defect disease.2. flow cytometry was used to detect the CD18 molecule.3.PCR sequencing of leukocyte surface and its parents' ITGB2 gene. Results: 1. clinical manifestation: all children were born from neonate with umbilical inflammation, followed by repeated skin mucosa and soft tissue infection (pneumonia, otitis media, thrush, gingivitis with early deciduous tooth removal), of which 2 cases (1 cases, 2) had navel With a history of delaying delay (21 days), 2 cases (1 cases, 3) had chronic skin infection and delayed wound healing (January). In 1 cases (1),.2. laboratory examination of premature premature infants had a significant increase in the total number of leukocytes in peripheral blood, mainly neutrophils, repeated infection, anemia, immunoglobulin Ig G, Ig A, Ig M increased, lymph nodes increased, lymph nodes increased, lymph nodes increased, lymph nodes increased, lymph nodes increased, lymph nodes increased, lymphatic M Cell classification, no obvious abnormal.3.CD18 flow analysis in NBT: the expression of CD18 molecules on the white cell surface was significantly reduced in children, and 2 was severe defects (CD18 in lymphocytes, neutrophils, mononuclear cells, 0,0.23%, 0), 1, and 3 were moderate defects (1:9.49%, 0.04%, 0.45%; 3:10.14%, 0.67%, 2.54%), but all parents CD18 expression 90%.4. ITGB2 gene analysis: 5 mutation sites (c.167_168ins GG, c.1884CA, c.533CT, c.817GC, c.1768TC) were found, of which 2 were new mutations (1 c.167-168ins GG, c.1884CA), and 5 carriers were found. Conclusion: severe skin and mucous membrane and soft tissue infection, especially with cord inflammation, umbilical cord shedding delay, wound healing delay, and repeated gums Inflammation with early deciduous teeth, the total number of white blood cells increased significantly, with neutrophils mainly, children with normal or elevated immunoglobulin need to be vigilant LAD-1. flow cytometry to detect the white cell surface CD18 molecules can quickly diagnose LAD-1, ITGB2 gene analysis is the gold standard of diagnosis.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R725.9

【參考文獻】

相關期刊論文 前7條

1 張超;陳國薇;楊玉萍;吳淑燕;劉箐;;活性氧調節(jié)單核細胞增生李斯特菌菌膜形成[J];食品科學;2013年23期

2 杜振蘭;;CD40-CD40L共刺激途徑在體液免疫和細胞免疫中的作用[J];細胞與分子免疫學雜志;2012年12期

3 陳同辛;金瑩瑩;;高IgM綜合征研究進展[J];實用兒科臨床雜志;2012年21期

4 楊學超;石云;鄒全明;;濾泡輔助T細胞的研究進展[J];重慶醫(yī)學;2011年23期

5 楊生海;殷宏;劉永生;張杰;;干擾素-γ研究進展[J];生物技術通報;2010年08期

6 王威;唐圣松;;吞噬細胞NADPH氧化酶活性的調控[J];國際病理科學與臨床雜志;2009年04期

7 冷麗麗;唐圣松;;NADPH氧化酶NOX家族的組織分布及生理功能[J];國際病理科學與臨床雜志;2008年01期

，

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