【正文】 四川大學(xué)臨床醫(yī)學(xué)碩士專業(yè)學(xué)位論文聲 明本人聲明所提交的學(xué)位論文是本人在導(dǎo)師的指導(dǎo)下進(jìn)行的研究工作及取得的研究成果。據(jù)我所知，除了文中特別加以標(biāo)注和致謝的地方外，論文中不包括其他人已經(jīng)發(fā)表或撰寫過的研究成果，也不包括為獲得四川大學(xué)或其他機(jī)構(gòu)的學(xué)位或證書而使用過的材料。與我一同工作的同志對(duì)本研究所作的任何貢獻(xiàn)均已在論文中做了明確的說明并表示謝意。本學(xué)位論文成果是本人在四川大學(xué)就讀期間取得的，論文成果歸四川大學(xué)所有，特此聲明。 論文作者簽名： 指導(dǎo)教師簽名：兒童急性淋巴細(xì)胞白血病IKZF1基因表達(dá)研究Expression of Active and DominantNegative IKZF1 Transcripts in Children with Acute Lymphoblastic Leukemia七年制研究生：夏 帆 導(dǎo) 師：高 舉 教授 指 導(dǎo) 教 師：袁粒星 副教授目 錄縮略詞表..........................................................................1中文摘要..........................................................................5英文摘要..........................................................................7前 言..........................................................................材料與方法......................................................................結(jié) 果..........................................................................討 論..........................................................................全文總結(jié)..........................................................................參考文獻(xiàn)..........................................................................綜 述..........................................................................致 謝..........................................................................縮 略 詞 表（按字母順序排序）縮寫英文中文全稱ALLacute lymphocytic leukemia急性淋巴細(xì)胞白血病BCRBcell antigen receptorB細(xì)胞抗原受體bpbase pair堿基對(duì)CCLGChildren’s Cancer and Leaukaemia Group 兒童腫瘤與白血病協(xié)作組CDcluster of differentiation分化抗體群cDNAplementary DNA互補(bǔ)DNAddH2Odoubledistilled water雙蒸水DEPCdiethypyrocarbonate焦碳酸二乙酯GAPDHglyceraldehyde3phosphate dehydrogenase甘油醛3磷酸脫氫酶GMPgranulomonocyte progenitors粒單核前體細(xì)胞HSChaemopoietic stem cell造血干細(xì)胞IKZF1(IK)Ikaros family zinc fingerIkaros 家族鋅指LMPPlymphoidprimed multipotent progenitors有淋巴細(xì)胞傾向的多潛能前體細(xì)胞mRNAMessenger RNA信使RNAODoptical density difference吸光度差Pprobability可能性PBSPhosphate Buffered Saline磷酸鹽緩沖液PCRPolymerase Chain Reaction聚合酶鏈?zhǔn)椒磻?yīng)Ph+Philadelphia Chromosome positive費(fèi)城染色體陽性RIribonuclease inhibitor核糖核酸酶抑制因子rpmrotation per minute每分鐘轉(zhuǎn)數(shù)TdTterminal deoxynucleotidyl transferase末端脫氧核苷酰轉(zhuǎn)移酶WBCWhite blood cells白細(xì)胞中文摘要目的 研究兒童ALL骨髓單個(gè)核細(xì)胞IKZF1(IK)基因可變剪接轉(zhuǎn)錄本表達(dá)情況，及其在各種臨床預(yù)后因素ALL亞組的表達(dá)譜(expression pattern)，探討其與兒童ALL發(fā)病和預(yù)后的可能關(guān)系。方法 采用巢式RTPCR方法，檢測我院兒童血液腫瘤科收治的43例ALL患兒骨髓單個(gè)核細(xì)胞和20例健康對(duì)照兒童外周血單個(gè)核細(xì)胞功能性IK和顯性負(fù)IK轉(zhuǎn)錄本表達(dá)情況，并經(jīng)DNA測序證實(shí)，統(tǒng)計(jì)分析不同預(yù)后因素ALL患兒IK可變剪接轉(zhuǎn)錄本的陽性表達(dá)率。結(jié)果 ALL患兒IK表達(dá)譜與健康對(duì)照兒童IK表達(dá)譜具有明顯差異。盡管兩組研究對(duì)象均表達(dá)IK各種轉(zhuǎn)錄本(包括功能性和顯性負(fù)IK轉(zhuǎn)錄本)，但對(duì)照組除1例外均表達(dá)功能性IK(尤其是IK2)，其表達(dá)水平明顯高于顯性負(fù)IK表達(dá)水平。而大多數(shù)ALL患兒表達(dá)顯性負(fù)IK，約44%的ALL患兒甚至僅較高水平表達(dá)一種或多種“顯性負(fù)”IK。1. ALL患兒IK2陽性表達(dá)率為51%，而對(duì)照組IK2陽性表達(dá)率為95%；ALL患兒和對(duì)照組功能性IK (IK1+IK2)陽性表達(dá)率分別為58%和95%，對(duì)照組陽性表達(dá)率均顯著高于ALL組(P)。2. 盡管從IK4陽性表達(dá)率而言，ALL顯性負(fù)IK4陽性表達(dá)率(%)顯著低于對(duì)照組(90%) (P)，但從圖3和圖4可以比較直觀地發(fā)現(xiàn)，ALL患兒IK4表達(dá)強(qiáng)度明顯高于對(duì)照組。其他2種顯性負(fù)IK陽性表達(dá)率組間無顯著性差異(P)。3. 標(biāo)危組、中危組和高危組ALL功能性IK(IK1+IK2)陽性表達(dá)率分別為80%、67%%；標(biāo)中危合并ALL組和高危ALL組功能性IK(IK1+IK2)陽性表達(dá)率分別為74%%，均具有統(tǒng)計(jì)學(xué)顯著差異（P）。單獨(dú)統(tǒng)計(jì)分析不同臨床危險(xiǎn)度ALL間各種顯性負(fù)IK陽性表達(dá)率無統(tǒng)計(jì)學(xué)顯著性差異。4. 潑尼松敏感ALL功能性IK(IK1+IK2)陽性表達(dá)率(71%)顯著高于潑尼松不敏感ALL(22%)(P)，但組間單一顯性負(fù)IK陽性表達(dá)率并無統(tǒng)計(jì)學(xué)顯著性差異。5. 除顯性負(fù)IK6外，初診WBC50180。109/L和初診WBC179。50180。109/L兩組ALL患兒IK陽性表達(dá)率無顯著性差異。結(jié)論 本文在國內(nèi)首次研究比較了ALL患兒骨髓單個(gè)核細(xì)胞和健康對(duì)照兒童外周血單個(gè)核細(xì)胞IK表達(dá)譜，及其與ALL各種預(yù)后因素的關(guān)系。初步研究結(jié)果顯示，兩組研究對(duì)象IK表達(dá)譜具有顯著差異，ALL患兒功能性IK陽性表達(dá)率顯著低于對(duì)照兒童，而且高危以及潑尼松不敏感ALL組功能性IK陽性表達(dá)率也顯著降低，提示IK可能在兒童ALL發(fā)病中發(fā)揮著重要作用，IK異常表達(dá)（功能性IK低表達(dá)和或顯性負(fù)IK高表達(dá)）可能是具有高危不良預(yù)后因素ALL的一個(gè)普遍特征。關(guān)鍵詞 急性淋巴細(xì)胞白血病(ALL)；Ikaros家族鋅指蛋白1( IKZF1)；巢式PCR；DNA測序；表達(dá)譜 (expression pattern)；兒童Expression of Active and DominantNegative IKZF1 Transcripts in Children with Acute Lymphoblastic LeukemiaPostgraduate: Xia Fan Tutor: Gao Ju, Prof of PediatricsABSTRACTOBJECTIVES: To determine the expression patterns of active and alternatively spliced dominantnegative Ikaros (IK) transcripts in children with acute lymphoblastic leukemia (ALL), and to explore the possible correlations between Ikaros expression and various prognostic risk factors in ALL.METHODS: The expression patterns of IK transcripts in bone marrow mononuclear cells and peripheral mononuclear cells, isolated from 43 children with ALL and 20 ageand sexmatched control children respectively, were determined by nested RTPCR. The identities of various functional /active and dominantnegative IK transcripts were judged by their corresponding electrophoretic positions on agarose gel and further confirmed by DNA sequencing. Expression positivities of every active and dominantnegative IK transcripts were pared between ALL group and control, and between ALL subgroups with different prognostic risk factors.RESULTS:1. Although both active and dominantnegative IK transcripts were expressed in mononuclear cells from ALL and normal control children, distinct overall expression patterns were documented in the two groups. High levels of expression of active IK (especially IK2) were detected in the majority of controls, while dominantnegative IKs were expressed in most of ALL cases, with 44% of ALL expressing exclusively one or more dominantnegative IK transcripts. 2. Expression of active IK transcripts was significantly lower in ALL (51% positivity for IK2, and 58% positivity for IK1+IK2) than in controls (95% positivity) (P).3. In terms of positive expression of active IK transcripts (IK1+IK2), there were statistically significant differences among the three risk groups of ALL, with much lower percentage of expression positivity in highrisk ALL (%) than that in standard risk (80%), medium risk (67%) or standard+medium risk (74%) ALL groups (P). Nevertheless, there existed no correlations between positive expression of each dominantnegative IK transcript and ALL risk categories. 4. Similarly, the ratio of positive expressions of active IK transcripts (IK1+IK2) was significantly higher in prednisone good responder (