發(fā)布時(shí)間：2018-05-31 09:16

  本文選題：原發(fā)性免疫缺陷病 + 調(diào)節(jié)性T細(xì)胞�。� 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：第一部分：IPEX新發(fā)突變一例分子與臨床特征研究 目的：探討1例X連鎖多內(nèi)分泌腺病、腸病伴免疫失調(diào)綜合征患兒FOXP3基因變異、蛋白表達(dá)水平、免疫表型及臨床特征。 方法：對我院收治的1例表現(xiàn)為早發(fā)性頑固性腹瀉、自身免疫現(xiàn)象、內(nèi)分泌功能異常、生長發(fā)育落后、IgE增高的疑似IPEX男性患兒外周血單個(gè)核細(xì)胞采用流式細(xì)胞儀檢測CD4+CD25+FOXP3+調(diào)節(jié)性T細(xì)胞比例和FOXP3蛋白表達(dá)，PCR法進(jìn)行外周血FOXP3基因擴(kuò)增及測序、比對分析，并與100例正常兒童FOXP3基因序列比較除外多態(tài)性以確定致病突變。 結(jié)果：該患兒CD4+CD25+FOXP3+調(diào)節(jié)性T細(xì)胞比例較正常對照明顯減低，F(xiàn)OXP3蛋白表達(dá)量與正常對照無顯著差異。經(jīng)基因分析確診為IPEX，其FOXP3基因第8號外顯子末位堿基錯(cuò)義突變(GA)，導(dǎo)致FOXP3蛋白323位氨基酸由谷氨酸替換為賴氨酸(Glu323Lys)，突變基因的轉(zhuǎn)錄產(chǎn)物中除了一部分為錯(cuò)義突變外，還形成了多種異常拼接體，部分為缺失第8號外顯子，部分同時(shí)缺失7、8號外顯子，另有部分完全或部分缺失5號外顯子及部分11號外顯子�；純耗赣H為該突變的攜帶者。100例正常兒童FOXP3基因相同位點(diǎn)未見變異。 結(jié)論：通過臨床、免疫學(xué)篩查、基因分析及流式細(xì)胞術(shù)，確診一例發(fā)生FOXP3多種不同異常拼接及錯(cuò)義突變的IPEX患兒，為此前未報(bào)到 的新發(fā)突變，，對早發(fā)頑固性腹瀉、濕疹、內(nèi)分泌功能異常伴自身免疫現(xiàn)象及不明腎臟損害嬰幼兒，應(yīng)考慮IPEX可能并進(jìn)行Treg流式細(xì)胞術(shù)快速篩查及FOXP3基因分析以最終確診。 第二部分：XLP-1型患者臨床、基因和蛋白表達(dá)分析 目的：探討SH2D1A基因突變所致XLP-1型患兒的臨床及分子特征，為臨床甄別和診斷此病提供信息。 方法：對15例臨床疑診為XLP-1型的男性患兒，收集歸納臨床資料和相關(guān)實(shí)驗(yàn)室檢查結(jié)果，采取患兒及其親屬新鮮外周血進(jìn)行流式細(xì)胞術(shù)檢測PBMC內(nèi)SAP蛋白的表達(dá)，提取外周血DNA和RNA，PCR法擴(kuò)增SH2D1A基因cDNA序列直接進(jìn)行測序。在NCBI基因庫比對目的序列，發(fā)現(xiàn)突變位點(diǎn)后擴(kuò)增相應(yīng)外顯子及或進(jìn)行TA克隆進(jìn)一步分析確定突變類型，同時(shí)對相關(guān)親屬進(jìn)行基因分析。 結(jié)果：通過對15例疑診XLP-1型患兒的基因和蛋白檢測，發(fā)現(xiàn)3例來自無血緣關(guān)系的不同家族的患兒存在SH2D1A基因突變，除外12例。3例突變均除外了基因多態(tài)性可能，其中2例為新發(fā)突變�；純�1的母親、外祖母及患兒2的母親為致病突變的攜帶者。行流式細(xì)胞術(shù)檢測的患兒1和2，SAP蛋白表達(dá)均明顯降低�；颊�1的母親SAP蛋白表達(dá)為雙峰，患者2的母親蛋白表達(dá)正常。 結(jié)論：本研究通過對患兒進(jìn)行臨床表現(xiàn)和免疫學(xué)初篩，同時(shí)借助基因序列分析和流式蛋白檢測，確診了3例SH2D1A基因不同突變的患者。對于符合XLP-1型臨床表現(xiàn)的患兒應(yīng)盡快進(jìn)行SAP蛋白檢測和SH2D1A基因分析。
[Abstract]:Part one: molecular and clinical characteristics of a new mutation of IPEX Objective: to investigate the FOXP3 gene variation, protein expression, immunophenotype and clinical features in a case of X-linked polyendocrine adenopathy and enteropathy with immune maladjustment syndrome. Methods: one patient with early onset refractory diarrhea, autoimmune phenomenon and abnormal endocrine function was treated in our hospital. Peripheral blood mononuclear cells (PBMC) of suspected male children with increased IPEX were amplified and sequenced by FOXP3 gene amplification and sequencing using flow cytometry to detect the proportion of CD4 CD25 FOXP3 regulatory T cells and the expression of FOXP3 protein. In order to identify the pathogenicity mutation, the FOXP3 gene sequence was compared with that of 100 normal children. Results: the percentage of regulatory T cells in CD4 CD25 FOXP3 was significantly lower than that in normal controls. There was no significant difference in the expression of FOXP3 protein between the two groups. It was confirmed by gene analysis that the missense mutation at the end of exon 8 of FOXP3 gene resulted in the amino acid substitution of FOXP3 protein 323 from glutamic acid to Glu323LysN. The transcriptional product of the mutant gene was missense mutation except for part of the transcriptional product. A variety of abnormal splicing bodies were formed, some of them were missing exon 8, some of them were deletion of exon 7,8 at the same time, and some of them were completely or partially deleted exon 5 and exon 11. The mother was the carrier of the mutation. There was no mutation in the same locus of the FOXP3 gene in the normal children. Conclusion: through clinical, immunological screening, gene analysis and flow cytometry, a case of IPEX with multiple abnormal splicing and missense mutation of FOXP3 was diagnosed. For early onset refractory diarrhea eczema abnormal endocrine function with autoimmune phenomenon and unknown kidney damage infants should consider the possibility of IPEX and carry out rapid screening of Treg flow cytometry and FOXP3 gene analysis to determine the final diagnosis. Part two: clinical, gene and protein expression in patients with type XLP-1 Objective: to investigate the clinical and molecular characteristics of XLP-1 type in children with SH2D1A gene mutation, and to provide information for clinical identification and diagnosis. Methods: 15 male children with suspected XLP-1 type were collected and summarized. The expression of SAP protein in PBMC was detected by flow cytometry in the fresh peripheral blood of the children and their relatives. The cDNA sequence of SH2D1A gene was amplified by DNA from peripheral blood and sequenced directly. After the target sequence of NCBI gene bank alignment was found, the corresponding exons were amplified and TA cloning was carried out to determine the mutation type, and the related relatives were analyzed. Results: by detecting the gene and protein of 15 children with suspected XLP-1 type, it was found that 3 children from different families without blood relationship had SH2D1A gene mutation, except 12 cases with SH2D1A gene mutation, all of them excluded the possibility of gene polymorphism. Two of them were new mutations. The mother of child 1, grandmother and mother of child 2 were carriers of the disease mutation. The expression of 1 and 2 SAP protein was significantly decreased by flow cytometry. The expression of SAP protein in the mother of patient 1 was bimodal, while the expression of protein in the mother of patient 2 was normal. Conclusion: in this study, three patients with different mutations of SH2D1A gene were diagnosed by screening their clinical manifestations, immunology, gene sequence analysis and flow protein detection. SAP protein detection and SH2D1A gene analysis should be carried out as soon as possible for children with XLP-1 type clinical manifestations.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R725.9

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相關(guān)期刊論文 前2條

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