儿童噬血细胞综合征的临床特征及新突变位点的致病机制研究
发布时间：2018-09-04 15:10
【摘要】：研究背景和目的噬血细胞综合征(Hemophagocytic lymphohistiocytosis,HLH)是儿科危重疾病,常常以不明原因发热起病,广谱抗生素治疗无效,病情进展迅速,有多脏器受损。没有及时和有效的治疗和支持,死亡率达100%。HLH的临床表现和实验室指标多样且缺乏特异性,目前HLH的诊断和治疗主要依据国际组织细胞协会制定的HLH-2004方案。然而该方案诊断准确敏感度不足,且不能判断疾病严重程度。HLH分为原发性和继发性两大类。原发性HLH是遗传性疾病,患者常常有家族史或者明确的基因突变。而继发性HLH的患者无基因缺陷,多继发于病毒感染、风湿免疫病或恶性肿瘤。原发性HLH有较高的复发率和预后差,异基因造血干细胞移植是唯一治愈的方法。明确原发性HLH的诊断对治疗的选择有重要指导意义。为了能提高儿童HLH的诊断和生存率、以及建立原发性HLH的诊断方法,进一步阐释HLH的发病机制,本课题进行了如下三个部分的研究:(1)儿童噬血细胞综合征的临床特征分析及基因突变情况调查;(2)原发性噬血细胞综合征诊断方法的研究;(3)噬血细胞综合征相关基因新突变位点的致病性机制研究。方法1儿童噬血细胞综合征的临床特征分析及基因突变情况调查1.1儿童噬血细胞综合征的临床特征分析:本研究收集了 2010年1月至2016年12月期间本院血液肿瘤科诊断为HLH的患儿,收集患儿的人口学特征,诊断时的临床症状和体征、常规实验室检测结果、血清Th1/Th2细胞因子和T/B/NK细胞比例等检测结果,以及生存情况。并进行统计分析,计量资料采用Mann-Whitney U-tests检验方法,计数资料采用χ2检验,多因素Cox回归模型筛选30天内死亡的独立预后因素。30天OS使用Kaplan-Meier评估和log-rank检验。1.2儿童噬血细胞综合征基因突变情况调查:本研究对诊断为HLH的患儿进行HLH相关基因测序;基因测序结果通过文献检索和数据库筛选,排除单核苷酸多态性位点。汇总测序结果,分析患儿基因突变情况;软件预测突变位点对蛋白功能影响;2原发性噬血细胞综合征诊断方法的研究:流式细胞术检测NK的HLH相关蛋白表达,包括:perforin、Munc13-4、syntaxin 11、Munc18-2、SAP、XIAP、Rab27a、AP3β1;流式细胞术检测NK细胞的脱颗粒功能,设置孵育效靶比和时间梯度筛选最适条件;流式细胞术检测NK细胞杀伤活性,对比CD33和CD147在K562表达表达,设置效靶比梯度筛选最适条件;建立三种实验方法的正常范围,并比较基因突变患儿、无基因突变患儿、健康儿童三组间的差异。3噬血细胞综合征相关基因新突变位点的致病机制研究3.1 UNC13D野生型和突变型慢病毒表达载体的构建:抽提患儿外周血RNA,逆转录成cDNA作为模板扩增目的基因;目的基因TA克隆,筛选正确的序列;通过酶切连接到慢病毒穿梭质粒上;抽提慢病毒穿梭质粒和包装质粒,进行慢病毒过表达载体包装,并采用有限稀释法检测慢病毒滴度。3.2突变型UNC13D对细胞毒T细胞(CTL)功能的影响:淋巴细胞分离液分离外周血单个核细胞,磁珠阴性分选分离CTL,加抗人CD3/CD28磁珠和重组IL-2培养扩增CTL;慢病毒感染CTL,使用荧光显微镜、流式细胞术和Western Blot法检测目的基因在CTL内表达;观察突变蛋白对CTL脱颗粒功能和杀伤活性的影响。结果1儿童噬血细胞综合征的临床和实验室检查特征分析及基因突变情况调查1.1儿童噬血细胞综合征的临床及实验检查特征分析:儿童HLH发病年龄集中在0-4岁,10岁以上发病的较少见;EB病毒是儿童HLH的最常见触发因素;本研究的患儿存活率为61.0%;诊断时患儿临床表现如下:均有高热、高血清铁蛋白、血二系以上降低和肝功能损害,骨髓噬血现象(96.5%)、肝脏肿大(83.2%)、低纤维蛋白原(76.4%)、脾脏肿大(67.5%)、高甘油三酯血症(48.7%)、呼吸系统受累(41.6%)、浅表淋巴结肿大(21.2%)、皮疹(18.6%)、消化系统症状(15.9%)、黄疸(7.1%)、中枢神经系统症状(6.2%);HLH患儿的INF-γ、IL-6、IL-10水平高于正常儿童(40.7(4.6-1477.5)pg/mL vs.3.4(1.4-14.6)pg/mL,P0.001;604.4(4.6-5000.0)pg/mLvs.3.0(1.7-8.2)pg/mL,P0.001;210.2(2.4-5000.0)pg/mLvs.3.7(1.0-11.8)pg/mL,P0.001),B 和 NK 细胞的比例低于正常儿童(12.1(3.0-45.5)%vs.22.2(15.7-32.3)%,P0.001;4.2(0.2-26.4)%vs.9.8(1.6-15.1)%,P0.001).Alb28.5g/L(HR=3.672,95%CI 1.281-10.533,P=0.015)、ChE≤4283.0 U/L(HR=15.714,95%CI 2.078-118.840,P=0.008)、IL-10456.0 pg/mL(HR=2.946,95%CI 1.135-7.744,P=0.027)是HLH患儿早期死亡的独立预后因素。与有0或1个危险因素患儿相比,有3个危险因素患儿的早期死亡风险增加54.17倍(HR=54.17,95%CI 7.28-403.17,P0.001),有2个危险因素患儿的早期死亡风险增加9.93倍(HR=9.93,95%CI 2.55-36.68,P=0.001)。1.2儿童噬血细胞综合征基因突变情况调查:共82例患儿进行了 HLH相关基因检测。40.2%(33/82)的患儿携带基因突变,4例为半合子,单等位基因突变21例,双等位基因突变4例,复合杂合突变4例;UNC13D和LYST是最常见受累基因,各占24%,其次为SXTBP2;共发现38个基因突变位点,22个为新发现的位点;4个为剪切位点突变,1个为深部内含子突变,1个为移码突变,3个为无义突变,29个为错义突变;错义突变中,突变预测13个位点致病性可能大。2原发性噬血细胞综合征诊断方法的研究:成功建立健康儿童NK细胞的HLH相关蛋白表达的参考值,HLH患儿的蛋白表达与健康儿童无明显区别,携带PRF1突变患儿的perforin表达显著低于不携带突变的患儿和健康儿童(P=0.009,P=0.021),携带SH2D1A突变的患儿的SAP蛋白显著低于不携带突变的患儿和健康儿童(P0.001,P0.001);成功建立流式细胞术检测NK细胞脱颗粒功能方法,1:1为最佳效靶比和3小时为最佳孵育时间,脱颗粒功能的参考范围为8.4-37.8%,携带脱颗粒相关基因突变的患儿的脱颗粒功能显著低于不携带基因突变患儿和健康儿童(9.3(0.5-29.5)%vs.17.5(4.9-33.8)%,P0.001;9.3(0.5-29.5)%vs.19.8(8.4-36.8)%,P0.001)。成功建立流式细胞术检测NK细胞杀伤CD147标记的K562的实验方法,10:1为最佳效靶比,NK细胞杀伤活性与NK细胞的比例相关(P=0.014,r=0.438),NK杀伤活性的正常范围为10.8-74.2%;HLH患儿急性期NK杀伤活性低于缓解期和健康儿童(2.9(0.6-7.7)%vs.7.5(2.8-25.0)%,P0.001;2.9(0.6-7.7)%vs 9.8(3.1-23.3)%,P0.001),携带 PRF1 和/或脱颗粒相关基因突变的患儿的NK细胞杀伤活性低于健康儿童(21.9(3.8-43.1)%vs.39.6(10.9-74.2)%,P=0.003)。3噬血细胞综合征相关基因新突变位点的致病机制研究3.1 UNC13D野生型和突变型慢病毒表达载体的构建:凝胶电泳和测序验证扩增的野生型和突变型目的片段正确,且成功连接到慢病毒穿梭载体;以293T细胞成功包装了野生型和突变型的慢病毒过表达载体,浓缩过的病毒滴度达108数量级以上。3.2突变型UNC13D对细胞毒T细胞功能的影响:淋巴细胞分离加磁珠阴性分选的CTL纯度达95%;抗人CD3CD28磁珠联合IL-2可以培养和扩增CTL,扩增倍数达120倍以上;野生型和突变型慢病毒过表达载体成功感染CTL,感染效率为10-20%,经puromycin筛选后可以达50%以上;流式细胞术和Western Blot法验证野生蛋白和突变蛋白在CTL中成功表达。脱颗粒三次结果分别为P1:Control为9.8,UNC13D-WT 为 13.8,UNC13D-Mut 为 8.2;P2:Control 为 9.1,UNC13D-WT为 16.0,UNC13D-Mut 为 6.7;P3:Control 为 8.3,UNC13D-WT 为 15.2,UNC13D-Mut为6.9。杀伤实验三次结果分别为P1:Control为59.7%,UNC13D-WT为97.4%,UNC13D-Mut 为 47.7%;P2:Control 为 48.5%,UNC13D-WT 为 93.8%,UNC13D-Mut为 48.2%;P3:Control 为 55.4%,UNC13D-WT 为 95.2%,UNC13D-Mut 为 44.2%。结论1.儿童HLH的主要发病年龄为0到4岁,10岁以上发病较少见,EBV是儿童HLH最常见的诱因。2.发热、血二系以上减少、高血清铁蛋白、肝功能损害、EBV阳性是儿童HLH的常见表现,出现这些表现的患儿需高度怀疑HLH,尽快完善HLH其他相关检查。3.血清IL-10 和 IFN-γ 水平可以用于早期诊断 HLH;Alb≤28.5g/L、ChE≤4283.0 U/L、IL-10≥456.0pg/mL是HLH患儿早期死亡的独立危险因素。4.40.2%的患儿携带基因突变,UNC13D、LYST是最常见的受累基因。大多数患儿为杂合突变,单独的基因检测不能做出原发性HLH的诊断。5.成功建立流式细胞术检测NK细胞的HLH相关蛋白、脱颗粒功能和杀伤活性实验。流式细胞术检测perforin和SAP低于正常值,提示存在PRF1和SH2D1A的基因突变。NK细胞脱颗粒功能和杀伤活性正常可以排除原发性HLH,低于正常需高度怀疑原发性HLH。6.UNC13D:c.2295_2298delGCAG,p.Glu765Aspfs*27 为致病型突变位点,该突变编码无功能蛋白。7.成功建立基于CTL的体外突变位点致病性验证方法。8.原发性HLH需基因检测联合蛋白表达、功能实验、突变位点验证来共同诊断。
[Abstract]:BACKGROUND AND OBJECTIVE Hemophagocytic lymphocyte syndrome (HLH) is a serious disease in pediatrics. It often starts with fever of unknown origin. Broad-spectrum antibiotics therapy is ineffective. The disease progresses rapidly with multiple organ damage. Without timely and effective treatment and support, the mortality rate of HLH is 100%. At present, the diagnosis and treatment of HLH are mainly based on the HLH-2004 protocol formulated by the International Association of Cells. However, the accuracy and sensitivity of the protocol are insufficient and the severity of the disease can not be judged. HLH can be divided into two categories: primary and secondary. Primary HLH is a genetic disease, and patients often have a family history or a clear family history. The primary HLH has a high recurrence rate and a poor prognosis. Allogeneic hematopoietic stem cell transplantation is the only cure method. The diagnosis of primary HLH is important for the choice of treatment. The diagnosis and survival rate of LH, and the establishment of a diagnostic method of primary HLH, further elucidate the pathogenesis of HLH, this topic carried out the following three parts of the study: (1) children's hemophagocytic syndrome clinical characteristics and gene mutation investigation; (2) primary hemophagocytic syndrome diagnosis methods; (3) hemophagocytic synthesis Pathogenic mechanism of new mutation sites of related genes. Methods 1 Clinical characteristics and gene mutation of hemophagocytic syndrome in children 1.1 Clinical characteristics of hemophagocytic syndrome in children 1. This study collected children diagnosed as HLH in our department of hematological oncology from January 2010 to December 2016. Demographic characteristics, clinical symptoms and signs at diagnosis, routine laboratory test results, serum Th1/Th2 cytokines and T/B/NK cell ratios, and survival conditions were analyzed. Mann-Whitney U-tests were used for measurement data, _2 test for counting data and multivariate Cox regression model for screening within 30 days. Independent prognostic factors for death. 30-day OS was assessed using Kaplan-Meier and log-rank tests. 1.2 Investigation of gene mutations in children with hemophagocytic syndrome: HLH-related gene sequencing was performed in children diagnosed with HLH; single nucleotide polymorphisms were excluded by literature search and database screening. Results: Analysis of gene mutations in children; Software predicted the impact of mutation sites on protein function; 2 Diagnostic methods of primary hemophagocytic syndrome: Flow cytometry detection of NK HL-related protein expression, including: perforin, Munc13-4, syntaxin 11, Munc18-2, SAP, XIAP, Rab27a, AP3 beta 1; Flow cytometry detection of NK cell degranulation function, Establish incubation target ratio and time gradient screening optimum conditions; Flow cytometry to detect NK cell killing activity, compare CD33 and CD147 expression in K562, set up the optimum conditions of target ratio gradient screening; Establish the normal range of three experimental methods, and compare the differences among three groups of children with gene mutation, children without gene mutation, healthy children. Pathogenic mechanism of new mutation sites of hemophagocytic syndrome-related genes 3.1 Construction of wild-type and mutant lentiviral expression vectors of UNC13D: RNA was extracted from peripheral blood of children, and reverse transcription cDNA was used as template to amplify the target gene; TA cloning of the target gene was used to screen the correct sequence; ligation of lentiviral shuttle plasmid by enzyme digestion; extraction of lentiviral shuttle plasmid; Lentivirus shuttle plasmids and packaged plasmids were packaged with lentivirus overexpression vectors. The effects of lentivirus titer 3.2 mutant UNC13D on cytotoxic T lymphocyte (CTL) function were detected by limited dilution method. Peripheral blood mononuclear cells (PBMC) were isolated from lymphocyte isolate, CTL was isolated by magnetic bead negative sorting, CTL was cultured with anti-human CD3/CD28 magnetic beads and recombinant IL-2. The expression of the target gene in CTL was detected by fluorescence microscopy, flow cytometry and Western Blot assay. The effects of the mutant protein on the degranulation function and killing activity of CTL were observed. Clinical and laboratory features of hematocyte syndrome: HLH in children aged 0-4 years old, 10 years old and older is rare; EB virus is the most common trigger of HLH in children; the survival rate of the children in this study was 61.0%; the clinical manifestations of the children at diagnosis are as follows: high fever, high serum ferritin, lower blood levels above the second line and liver function. Can damage, bone marrow hemophagocytosis (96.5%), liver enlargement (83.2%), hypofibrinogen (76.4%), splenomegaly (67.5%), hypertriglyceridemia (48.7%), respiratory system involvement (41.6%), superficial lymphadenopathy (21.2%), rash (18.6%), digestive system symptoms (15.9%), jaundice (7.1%), central nervous system symptoms (6.2%) HLH children's INF-gamma, IL-6, IL-10. 姘村钩楂樹簬姝ｅ父鍎跨(40.7(4.6-1477.5)pg/mL vs.3.4(1.4-14.6)pg/mL,P0.001;604.4(4.6-5000.0)pg/mLvs.3.0(1.7-8.2)pg/mL,P0.001;210.2(2.4-5000.0)pg/mLvs.3.7(1.0-11.8)pg/mL,P0.001),B 鍜，
本文编号：2222534
[Abstract]:BACKGROUND AND OBJECTIVE Hemophagocytic lymphocyte syndrome (HLH) is a serious disease in pediatrics. It often starts with fever of unknown origin. Broad-spectrum antibiotics therapy is ineffective. The disease progresses rapidly with multiple organ damage. Without timely and effective treatment and support, the mortality rate of HLH is 100%. At present, the diagnosis and treatment of HLH are mainly based on the HLH-2004 protocol formulated by the International Association of Cells. However, the accuracy and sensitivity of the protocol are insufficient and the severity of the disease can not be judged. HLH can be divided into two categories: primary and secondary. Primary HLH is a genetic disease, and patients often have a family history or a clear family history. The primary HLH has a high recurrence rate and a poor prognosis. Allogeneic hematopoietic stem cell transplantation is the only cure method. The diagnosis of primary HLH is important for the choice of treatment. The diagnosis and survival rate of LH, and the establishment of a diagnostic method of primary HLH, further elucidate the pathogenesis of HLH, this topic carried out the following three parts of the study: (1) children's hemophagocytic syndrome clinical characteristics and gene mutation investigation; (2) primary hemophagocytic syndrome diagnosis methods; (3) hemophagocytic synthesis Pathogenic mechanism of new mutation sites of related genes. Methods 1 Clinical characteristics and gene mutation of hemophagocytic syndrome in children 1.1 Clinical characteristics of hemophagocytic syndrome in children 1. This study collected children diagnosed as HLH in our department of hematological oncology from January 2010 to December 2016. Demographic characteristics, clinical symptoms and signs at diagnosis, routine laboratory test results, serum Th1/Th2 cytokines and T/B/NK cell ratios, and survival conditions were analyzed. Mann-Whitney U-tests were used for measurement data, _2 test for counting data and multivariate Cox regression model for screening within 30 days. Independent prognostic factors for death. 30-day OS was assessed using Kaplan-Meier and log-rank tests. 1.2 Investigation of gene mutations in children with hemophagocytic syndrome: HLH-related gene sequencing was performed in children diagnosed with HLH; single nucleotide polymorphisms were excluded by literature search and database screening. Results: Analysis of gene mutations in children; Software predicted the impact of mutation sites on protein function; 2 Diagnostic methods of primary hemophagocytic syndrome: Flow cytometry detection of NK HL-related protein expression, including: perforin, Munc13-4, syntaxin 11, Munc18-2, SAP, XIAP, Rab27a, AP3 beta 1; Flow cytometry detection of NK cell degranulation function, Establish incubation target ratio and time gradient screening optimum conditions; Flow cytometry to detect NK cell killing activity, compare CD33 and CD147 expression in K562, set up the optimum conditions of target ratio gradient screening; Establish the normal range of three experimental methods, and compare the differences among three groups of children with gene mutation, children without gene mutation, healthy children. Pathogenic mechanism of new mutation sites of hemophagocytic syndrome-related genes 3.1 Construction of wild-type and mutant lentiviral expression vectors of UNC13D: RNA was extracted from peripheral blood of children, and reverse transcription cDNA was used as template to amplify the target gene; TA cloning of the target gene was used to screen the correct sequence; ligation of lentiviral shuttle plasmid by enzyme digestion; extraction of lentiviral shuttle plasmid; Lentivirus shuttle plasmids and packaged plasmids were packaged with lentivirus overexpression vectors. The effects of lentivirus titer 3.2 mutant UNC13D on cytotoxic T lymphocyte (CTL) function were detected by limited dilution method. Peripheral blood mononuclear cells (PBMC) were isolated from lymphocyte isolate, CTL was isolated by magnetic bead negative sorting, CTL was cultured with anti-human CD3/CD28 magnetic beads and recombinant IL-2. The expression of the target gene in CTL was detected by fluorescence microscopy, flow cytometry and Western Blot assay. The effects of the mutant protein on the degranulation function and killing activity of CTL were observed. Clinical and laboratory features of hematocyte syndrome: HLH in children aged 0-4 years old, 10 years old and older is rare; EB virus is the most common trigger of HLH in children; the survival rate of the children in this study was 61.0%; the clinical manifestations of the children at diagnosis are as follows: high fever, high serum ferritin, lower blood levels above the second line and liver function. Can damage, bone marrow hemophagocytosis (96.5%), liver enlargement (83.2%), hypofibrinogen (76.4%), splenomegaly (67.5%), hypertriglyceridemia (48.7%), respiratory system involvement (41.6%), superficial lymphadenopathy (21.2%), rash (18.6%), digestive system symptoms (15.9%), jaundice (7.1%), central nervous system symptoms (6.2%) HLH children's INF-gamma, IL-6, IL-10. 姘村钩楂樹簬姝ｅ父鍎跨(40.7(4.6-1477.5)pg/mL vs.3.4(1.4-14.6)pg/mL,P0.001;604.4(4.6-5000.0)pg/mLvs.3.0(1.7-8.2)pg/mL,P0.001;210.2(2.4-5000.0)pg/mLvs.3.7(1.0-11.8)pg/mL,P0.001),B 鍜，
本文编号：2222534
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