【摘要】：研究背景和目的噬血細(xì)胞綜合征(Hemophagocytic lymphohistiocytosis,HLH)是兒科危重疾病,常常以不明原因發(fā)熱起病,廣譜抗生素治療無(wú)效,病情進(jìn)展迅速,有多臟器受損。沒(méi)有及時(shí)和有效的治療和支持,死亡率達(dá)100%。HLH的臨床表現(xiàn)和實(shí)驗(yàn)室指標(biāo)多樣且缺乏特異性,目前HLH的診斷和治療主要依據(jù)國(guó)際組織細(xì)胞協(xié)會(huì)制定的HLH-2004方案。然而該方案診斷準(zhǔn)確敏感度不足,且不能判斷疾病嚴(yán)重程度。HLH分為原發(fā)性和繼發(fā)性兩大類。原發(fā)性HLH是遺傳性疾病,患者常常有家族史或者明確的基因突變。而繼發(fā)性HLH的患者無(wú)基因缺陷,多繼發(fā)于病毒感染、風(fēng)濕免疫病或惡性腫瘤。原發(fā)性HLH有較高的復(fù)發(fā)率和預(yù)后差,異基因造血干細(xì)胞移植是唯一治愈的方法。明確原發(fā)性HLH的診斷對(duì)治療的選擇有重要指導(dǎo)意義。為了能提高兒童HLH的診斷和生存率、以及建立原發(fā)性HLH的診斷方法,進(jìn)一步闡釋HLH的發(fā)病機(jī)制,本課題進(jìn)行了如下三個(gè)部分的研究:(1)兒童噬血細(xì)胞綜合征的臨床特征分析及基因突變情況調(diào)查;(2)原發(fā)性噬血細(xì)胞綜合征診斷方法的研究;(3)噬血細(xì)胞綜合征相關(guān)基因新突變位點(diǎn)的致病性機(jī)制研究。方法1兒童噬血細(xì)胞綜合征的臨床特征分析及基因突變情況調(diào)查1.1兒童噬血細(xì)胞綜合征的臨床特征分析:本研究收集了 2010年1月至2016年12月期間本院血液腫瘤科診斷為HLH的患兒,收集患兒的人口學(xué)特征,診斷時(shí)的臨床癥狀和體征、常規(guī)實(shí)驗(yàn)室檢測(cè)結(jié)果、血清Th1/Th2細(xì)胞因子和T/B/NK細(xì)胞比例等檢測(cè)結(jié)果,以及生存情況。并進(jìn)行統(tǒng)計(jì)分析,計(jì)量資料采用Mann-Whitney U-tests檢驗(yàn)方法,計(jì)數(shù)資料采用χ2檢驗(yàn),多因素Cox回歸模型篩選30天內(nèi)死亡的獨(dú)立預(yù)后因素。30天OS使用Kaplan-Meier評(píng)估和log-rank檢驗(yàn)。1.2兒童噬血細(xì)胞綜合征基因突變情況調(diào)查:本研究對(duì)診斷為HLH的患兒進(jìn)行HLH相關(guān)基因測(cè)序;基因測(cè)序結(jié)果通過(guò)文獻(xiàn)檢索和數(shù)據(jù)庫(kù)篩選,排除單核苷酸多態(tài)性位點(diǎn)。匯總測(cè)序結(jié)果,分析患兒基因突變情況;軟件預(yù)測(cè)突變位點(diǎn)對(duì)蛋白功能影響;2原發(fā)性噬血細(xì)胞綜合征診斷方法的研究:流式細(xì)胞術(shù)檢測(cè)NK的HLH相關(guān)蛋白表達(dá),包括:perforin、Munc13-4、syntaxin 11、Munc18-2、SAP、XIAP、Rab27a、AP3β1;流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞的脫顆粒功能,設(shè)置孵育效靶比和時(shí)間梯度篩選最適條件;流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞殺傷活性,對(duì)比CD33和CD147在K562表達(dá)表達(dá),設(shè)置效靶比梯度篩選最適條件;建立三種實(shí)驗(yàn)方法的正常范圍,并比較基因突變患兒、無(wú)基因突變患兒、健康兒童三組間的差異。3噬血細(xì)胞綜合征相關(guān)基因新突變位點(diǎn)的致病機(jī)制研究3.1 UNC13D野生型和突變型慢病毒表達(dá)載體的構(gòu)建:抽提患兒外周血RNA,逆轉(zhuǎn)錄成cDNA作為模板擴(kuò)增目的基因;目的基因TA克隆,篩選正確的序列;通過(guò)酶切連接到慢病毒穿梭質(zhì)粒上;抽提慢病毒穿梭質(zhì)粒和包裝質(zhì)粒,進(jìn)行慢病毒過(guò)表達(dá)載體包裝,并采用有限稀釋法檢測(cè)慢病毒滴度。3.2突變型UNC13D對(duì)細(xì)胞毒T細(xì)胞(CTL)功能的影響:淋巴細(xì)胞分離液分離外周血單個(gè)核細(xì)胞,磁珠陰性分選分離CTL,加抗人CD3/CD28磁珠和重組IL-2培養(yǎng)擴(kuò)增CTL;慢病毒感染CTL,使用熒光顯微鏡、流式細(xì)胞術(shù)和Western Blot法檢測(cè)目的基因在CTL內(nèi)表達(dá);觀察突變蛋白對(duì)CTL脫顆粒功能和殺傷活性的影響。結(jié)果1兒童噬血細(xì)胞綜合征的臨床和實(shí)驗(yàn)室檢查特征分析及基因突變情況調(diào)查1.1兒童噬血細(xì)胞綜合征的臨床及實(shí)驗(yàn)檢查特征分析:兒童HLH發(fā)病年齡集中在0-4歲,10歲以上發(fā)病的較少見(jiàn);EB病毒是兒童HLH的最常見(jiàn)觸發(fā)因素;本研究的患兒存活率為61.0%;診斷時(shí)患兒臨床表現(xiàn)如下:均有高熱、高血清鐵蛋白、血二系以上降低和肝功能損害,骨髓噬血現(xiàn)象(96.5%)、肝臟腫大(83.2%)、低纖維蛋白原(76.4%)、脾臟腫大(67.5%)、高甘油三酯血癥(48.7%)、呼吸系統(tǒng)受累(41.6%)、淺表淋巴結(jié)腫大(21.2%)、皮疹(18.6%)、消化系統(tǒng)癥狀(15.9%)、黃疸(7.1%)、中樞神經(jīng)系統(tǒng)癥狀(6.2%);HLH患兒的INF-γ、IL-6、IL-10水平高于正常兒童(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 細(xì)胞的比例低于正常兒童(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患兒早期死亡的獨(dú)立預(yù)后因素。與有0或1個(gè)危險(xiǎn)因素患兒相比,有3個(gè)危險(xiǎn)因素患兒的早期死亡風(fēng)險(xiǎn)增加54.17倍(HR=54.17,95%CI 7.28-403.17,P0.001),有2個(gè)危險(xiǎn)因素患兒的早期死亡風(fēng)險(xiǎn)增加9.93倍(HR=9.93,95%CI 2.55-36.68,P=0.001)。1.2兒童噬血細(xì)胞綜合征基因突變情況調(diào)查:共82例患兒進(jìn)行了 HLH相關(guān)基因檢測(cè)。40.2%(33/82)的患兒攜帶基因突變,4例為半合子,單等位基因突變21例,雙等位基因突變4例,復(fù)合雜合突變4例;UNC13D和LYST是最常見(jiàn)受累基因,各占24%,其次為SXTBP2;共發(fā)現(xiàn)38個(gè)基因突變位點(diǎn),22個(gè)為新發(fā)現(xiàn)的位點(diǎn);4個(gè)為剪切位點(diǎn)突變,1個(gè)為深部?jī)?nèi)含子突變,1個(gè)為移碼突變,3個(gè)為無(wú)義突變,29個(gè)為錯(cuò)義突變;錯(cuò)義突變中,突變預(yù)測(cè)13個(gè)位點(diǎn)致病性可能大。2原發(fā)性噬血細(xì)胞綜合征診斷方法的研究:成功建立健康兒童NK細(xì)胞的HLH相關(guān)蛋白表達(dá)的參考值,HLH患兒的蛋白表達(dá)與健康兒童無(wú)明顯區(qū)別,攜帶PRF1突變患兒的perforin表達(dá)顯著低于不攜帶突變的患兒和健康兒童(P=0.009,P=0.021),攜帶SH2D1A突變的患兒的SAP蛋白顯著低于不攜帶突變的患兒和健康兒童(P0.001,P0.001);成功建立流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞脫顆粒功能方法,1:1為最佳效靶比和3小時(shí)為最佳孵育時(shí)間,脫顆粒功能的參考范圍為8.4-37.8%,攜帶脫顆粒相關(guān)基因突變的患兒的脫顆粒功能顯著低于不攜帶基因突變患兒和健康兒童(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)。成功建立流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞殺傷CD147標(biāo)記的K562的實(shí)驗(yàn)方法,10:1為最佳效靶比,NK細(xì)胞殺傷活性與NK細(xì)胞的比例相關(guān)(P=0.014,r=0.438),NK殺傷活性的正常范圍為10.8-74.2%;HLH患兒急性期NK殺傷活性低于緩解期和健康兒童(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),攜帶 PRF1 和/或脫顆粒相關(guān)基因突變的患兒的NK細(xì)胞殺傷活性低于健康兒童(21.9(3.8-43.1)%vs.39.6(10.9-74.2)%,P=0.003)。3噬血細(xì)胞綜合征相關(guān)基因新突變位點(diǎn)的致病機(jī)制研究3.1 UNC13D野生型和突變型慢病毒表達(dá)載體的構(gòu)建:凝膠電泳和測(cè)序驗(yàn)證擴(kuò)增的野生型和突變型目的片段正確,且成功連接到慢病毒穿梭載體;以293T細(xì)胞成功包裝了野生型和突變型的慢病毒過(guò)表達(dá)載體,濃縮過(guò)的病毒滴度達(dá)108數(shù)量級(jí)以上。3.2突變型UNC13D對(duì)細(xì)胞毒T細(xì)胞功能的影響:淋巴細(xì)胞分離加磁珠陰性分選的CTL純度達(dá)95%;抗人CD3CD28磁珠聯(lián)合IL-2可以培養(yǎng)和擴(kuò)增CTL,擴(kuò)增倍數(shù)達(dá)120倍以上;野生型和突變型慢病毒過(guò)表達(dá)載體成功感染CTL,感染效率為10-20%,經(jīng)puromycin篩選后可以達(dá)50%以上;流式細(xì)胞術(shù)和Western Blot法驗(yàn)證野生蛋白和突變蛋白在CTL中成功表達(dá)。脫顆粒三次結(jié)果分別為P1:Control為9.8,UNC13D-WT 為 13.8,UNC13D-Mut 為 8.2;P2:Control 為 9.1,UNC13D-WT為 16.0,UNC13D-Mut 為 6.7;P3:Control 為 8.3,UNC13D-WT 為 15.2,UNC13D-Mut為6.9。殺傷實(shí)驗(yàn)三次結(jié)果分別為P1:Control為59.7%,UNC13D-WT為97.4%,UNC13D-Mut 為 47.7%;P2:Control 為 48.5%,UNC13D-WT 為 93.8%,UNC13D-Mut為 48.2%;P3:Control 為 55.4%,UNC13D-WT 為 95.2%,UNC13D-Mut 為 44.2%。結(jié)論1.兒童HLH的主要發(fā)病年齡為0到4歲,10歲以上發(fā)病較少見(jiàn),EBV是兒童HLH最常見(jiàn)的誘因。2.發(fā)熱、血二系以上減少、高血清鐵蛋白、肝功能損害、EBV陽(yáng)性是兒童HLH的常見(jiàn)表現(xiàn),出現(xiàn)這些表現(xiàn)的患兒需高度懷疑HLH,盡快完善HLH其他相關(guān)檢查。3.血清IL-10 和 IFN-γ 水平可以用于早期診斷 HLH;Alb≤28.5g/L、ChE≤4283.0 U/L、IL-10≥456.0pg/mL是HLH患兒早期死亡的獨(dú)立危險(xiǎn)因素。4.40.2%的患兒攜帶基因突變,UNC13D、LYST是最常見(jiàn)的受累基因。大多數(shù)患兒為雜合突變,單獨(dú)的基因檢測(cè)不能做出原發(fā)性HLH的診斷。5.成功建立流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞的HLH相關(guān)蛋白、脫顆粒功能和殺傷活性實(shí)驗(yàn)。流式細(xì)胞術(shù)檢測(cè)perforin和SAP低于正常值,提示存在PRF1和SH2D1A的基因突變。NK細(xì)胞脫顆粒功能和殺傷活性正�？梢耘懦l(fā)性HLH,低于正常需高度懷疑原發(fā)性HLH。6.UNC13D:c.2295_2298delGCAG,p.Glu765Aspfs*27 為致病型突變位點(diǎn),該突變編碼無(wú)功能蛋白。7.成功建立基于CTL的體外突變位點(diǎn)致病性驗(yàn)證方法。8.原發(fā)性HLH需基因檢測(cè)聯(lián)合蛋白表達(dá)、功能實(shí)驗(yàn)、突變位點(diǎn)驗(yàn)證來(lái)共同診斷。
[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. 姘村鉤楂樹(shù)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，

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