两种白细胞功能异常疾病的临床分子特征及机制研究

发布时间：2018-06-25 19:15

本文选题：慢性肉芽肿病 + 临床特征　；参考：《重庆医科大学》2017年硕士论文

【摘要】：第一部分慢性肉芽肿病患儿临床与分子特征研究目的:探讨26例慢性肉芽肿病(CGD)患儿的临床特征,实验室检查结果及基因突变特征。方法:1.收集2015年6月-2017年2月在重庆医科大学附属儿童医院收治的26例CGD患儿临床资料和实验室检查结果并分析总结。2.流式细胞术分析患儿中性粒细胞呼吸爆发功能,并计算中性粒细胞氧化指数NOI(刺激后平均荧光强度/刺激前平均荧光强度)。3.PCR直接测序法分析患儿CGD相关基因(CYBB、CYBA、NCF1、NCF2)。结果:1.一般情况:26例患儿均为男性,15例(57.7%)来自我国西南地区,平均起病年龄2.3月,平均诊断年龄1.4岁,14例(53.8%)有家族史(男性亲属早年夭折)。2.临床表现:26例患儿均有反复发热,肺炎(24例,92.3%)、腹泻(13例,50%)、皮肤黏膜及皮下组织感染(14例,53.8%)、肝脾肿大、肝功能异常(12例,46.2%)为本组患儿最常见的临床表现。21例接种卡介苗的患儿中14例(66.7%)发生BCG接种异常反应(卡疤化脓、BCG淋巴结炎、肺结核)。此外,3例(11.5%)发生肝脓肿,2例(7.7%)常患尿路感染3.实验室检查:本组患儿多出现外周血白细胞总数增高,以中性粒细胞为主,多有CRP增高。淋巴细胞分类未见明显异常,免疫球蛋白正常或代偿性增高,其中9例(34.6%)出现Ig E明显增高。除2例患儿NBT正常外,其余21例(80.8%)NBT均明显降低,多数PMA刺激后为0。仅50%患儿找到致病病原菌。4.呼吸爆发试验及基因突变分析:患儿NOI多数低于2(1.365±0.1018),明显低于正常值(正常人100),患儿母亲NOI正常或低于正常值;21例(80.8%)患儿发现CYBB基因突变,1例(3.8%)CYBA突变,4例(15.4%)尚未明确致病基因,发现15名XLR-CGD携带者。结论:CGD患儿多于生后即发生严重的细菌及真菌感染,患儿伴有BCG接种异常反应及男性亲属早年夭折家族史,白细胞总数显著增高,以中性粒细胞为主,NBT异常,免疫球蛋白正常或升高,则需警惕CGD。流式细胞术检测中性粒细胞呼吸爆发功能可快速诊断CGD,基因检测可进一步确诊患者、发现携带者,有产前诊断意义。第二部分慢性肉芽肿病患儿记忆B细胞减少的相关因素研究目的:探讨CGD患儿是否存在外周血记忆B细胞(MBC)减少的现象,寻找其减少的原因;并探索IFN-γ治疗CGD患儿,减少反复感染的可能机制,为临床运用IFN-γ治疗和预防反复感染提供理论依据。方法:1.流式细胞术检测CGD患儿MBC百分比、CD4+T细胞表面CD40L表达量、Tfh细胞及其表面PD-1百分比。2.不同浓度s CD40L(1μg/ml、1.5μg/ml、2μg/ml)与正常儿童外周血PBMC共同刺激培养24h、36h、48h,流式细胞术检测MBC数量的变化。3.分别用不同浓度的外源性H2O2(10μmol/L、30μmol/L)、IFN-γ(2000U/ml、3000U/ml)与CGD患儿外周血PBMC共同刺激培养24h,流式细胞术检测MBC数量的变化。结果:1.CGD患儿MBC百分比(1.267%±0.1416%)较健康对照儿童(2.824%±0.27%)明显降低(P0.0001)。2.CGD患儿CD4+T细胞表面CD40L百分比(8.186%±2.736%)较健康对照儿童(46.43%±4.619%)明显降低(P0.0001)。3.CGD患儿外周血Tfh细胞(5.625%±0.5005%)与健康儿童(6.269%±1.177%)无显著差异(P=0.5614)。PD-1百分比(9.039%±1.984%)与健康儿童(18.26%±10.11%)无显著差异(P=0.7546)。4.外源性补充s CD40L不能促进健康儿童MBC增高,MBC百分比随培养时间延长逐渐降低(P=0.0028)。5.外源性补充H2O2、IFN-γ对CGD患儿MBC无明显刺激作用,相反,MBC百分比随培养时间延长逐渐降低(P0.0001)。结论:CGD患儿外周血MBC减少可能是患儿反复感染的原因,MBC减少可能与CD4+T细胞表面CD40L表达减少相关,虽然CGD患儿外周血Tfh细胞数量正常,但仍需进一步分析Tfh细胞表面CD40L表达情况以明确Tfh细胞功能是否正常。IFN-γ并不通过刺激CGD患儿外周血MBC增殖发挥治疗作用。第三部分3例白细胞黏附分子缺陷病Ι型的临床及分子特征分析目的:探讨3例白细胞黏附分子缺陷病Ι型(LAD-1)患儿的临床特征和CD18蛋白表达异常及基因突变特征。方法:1.总结3例患儿临床资料,常规免疫学筛查除外常见原发性免疫缺陷病。2.流式细胞术检测白细胞表面CD18分子。3.PCR测序分析患儿及其父母ITGB2基因。结果:1.临床表现:患儿均自新生儿起以脐炎起病,之后经历反复皮肤黏膜及软组织感染(肺炎、中耳炎、鹅口疮、牙龈炎伴乳牙早脱),其中2例(例1、例2)有脐带脱落延迟史(21天),2例(例1、例3)有慢性皮肤感染及伤口愈合延迟(1月),1例(例1)家族中有早年夭折患儿。2.实验室检查:患儿均有外周血白细胞总数显著增高,以中性粒细胞为主,因反复感染,常伴有贫血,免疫球蛋白Ig G、Ig A、Ig M增高,淋巴细胞分类、NBT无明显异常。3.CD18流式分析:患儿白细胞表面CD18分子表达明显降低,例2为重度缺陷(CD18在淋巴细胞、中性粒细胞、单核细胞上分别为0、0.23%、0),例1、例3为中度缺陷(例1:9.49%、0.04%、0.45%;例3:10.14%、0.67%、2.54%),但所有父母CD18表达均90%。4.ITGB2基因分析:发现5种突变位点(c.167_168ins GG、c.1884CA、c.533CT、c.817GC、c.1768TC),其中2种为新型突变(例1 c.167-168ins GG、c.1884CA),并发现5名携带者。结论:自幼反复严重皮肤黏膜及软组织感染,尤其伴有脐炎、脐带脱落延迟,伤口愈合延迟,反复牙龈炎伴乳牙早脱,白细胞总数显著增高,以中性粒细胞为主,伴免疫球蛋白正常或升高的患儿需警惕LAD-1。流式细胞术检测白细胞表面CD18分子可快速诊断LAD-1,ITGB2基因分析是诊断的金标准。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.9

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