目的：1、建立自然殺傷性細(xì)胞及細(xì)胞毒性T細(xì)胞表面溶酶體相關(guān)膜蛋白質(zhì)1（LAMP-1，CD107α）表達(dá)流式細(xì)胞術(shù)檢測(cè)方法；2、探討其對(duì)自然殺傷性（NK）細(xì)胞與細(xì)胞毒性T淋巴細(xì)胞（CTL）細(xì)胞毒功能及相關(guān)性疾病的篩查價(jià)值。方法:收集疑診Chediak-Higashi綜合征（Chediak-HigashiSyndrome，CHS）患兒3例、噬血淋巴組織細(xì)胞增生癥（HLH）患兒3例及10例健康對(duì)照兒童外周血，提取外周血DNA及RNA，PCR或RT-PCR擴(kuò)增目的基因，測(cè)序分析基因背景。外周血單個(gè)核細(xì)胞（PBMC）以重組人白介素受體2（IL-2）刺激過夜，再以植物凝集素（PHA）或T細(xì)胞刺激劑Anti-CD3活化刺激細(xì)胞2h后，流式細(xì)胞術(shù)分析NK細(xì)胞及CTL CD107α表達(dá)頻率及強(qiáng)度。結(jié)果：10例健康兒童PBMC刺激前后NK細(xì)胞[（0.27±0.07）%vs.（5.80±2.83）%，P<0.05]及CTL[（0.18±0.07）%vs.（4.47±2.36）%，P<0.05]表面CD107α表達(dá)頻率均顯著升高，其平均熒光強(qiáng)度（MFI）亦顯著升高。與10例正常兒童比較，3例疑似CHS患兒外周血PBMC經(jīng)Anti-CD3活化后CTL表面CD107α表達(dá)無(wú)明顯上升（0.3%、0.9%、0.2%）,對(duì)照組（4.47±2.36）%；PHA刺激后兩例患兒NK細(xì)胞表面CD107α表達(dá)頻率亦無(wú)明顯變化（0.5%、0.6%），對(duì)照組（5.80±2.83）%，其MFI變化趨勢(shì)與CD107α表達(dá)率類似。3例HLH患兒NK細(xì)胞（3.3%、4.1%、2.7%），對(duì)照組（5.80±2.83）%；CTL（2.8%、1.7%、1.9%），對(duì)照組（4.47±2.36）%；表面CD107α刺激前后其表達(dá)頻率及MFI與正常對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義。3例疑似CHS患兒中兩例患兒LYST基因突變分別為（c.5411-5414del TTTC，L1741fsX1758和c.7975C>T，R2596X）和（c.4863G>A, R1563H和c.5392-5393delAA, E1739fsX1756），確診為CHS綜合征，另一例基因診斷尚未明確。3例HLH患兒PRF、MUNC13-4及STX11基因檢查結(jié)果均未發(fā)現(xiàn)突變。結(jié)論：成功建立CD107α表達(dá)流式細(xì)胞檢測(cè)方法，并在CHS中驗(yàn)證，該方法可能用作細(xì)胞毒功能的篩查方法，為后續(xù)基因確診及發(fā)現(xiàn)新的致病基因奠定基礎(chǔ)。

 

【文章來(lái)源】：重慶醫(yī)科大學(xué)重慶市

 

【文章頁(yè)數(shù)】：54 頁(yè)

 

【學(xué)位級(jí)別】：碩士

 

文章目錄

英漢縮略語(yǔ)名詞對(duì)照

中文摘要

英文摘要

前言

1 材料和方法

2 結(jié)果

3 討論

參考文獻(xiàn)

全文總結(jié)

文獻(xiàn)綜述

    參考文獻(xiàn)

致謝

攻讀碩士學(xué)位期間發(fā)表的論文

 

 

參考文獻(xiàn)

 

國(guó)際期刊論文

 

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