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細(xì)胞因子檢測(cè)技術(shù)

時(shí)間:2011/10/13閱讀:1010
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乾思 生物編輯整理: 教 師:XXX 實(shí)驗(yàn)類型:基礎(chǔ) 學(xué)  時(shí):8 (參考) 內(nèi)  容: 一、實(shí)驗(yàn)?zāi)康模?1.以檢測(cè)IL-2為例,使學(xué)生掌握一定的細(xì)胞培養(yǎng)技術(shù)。 2.使學(xué)生掌握一定的細(xì)胞因子檢測(cè)的一些主要方法。 3.掌握免疫細(xì)胞分離的主要技術(shù)和方法。 二、實(shí)驗(yàn)原理: 檢測(cè)細(xì)胞因子對(duì)于闡明機(jī)體免疫應(yīng)答機(jī)制及相關(guān)疾病的發(fā)生、發(fā)展規(guī)律和臨床治療都有重要意義。細(xì)胞因子的檢測(cè)主要分為免疫學(xué)、生物學(xué)和分子生物學(xué)三種檢測(cè)方法。 生物學(xué)方法檢測(cè)細(xì)胞因子活性,其原理時(shí)根據(jù)細(xì)胞因子對(duì)特定的依賴性細(xì)胞株(即靶細(xì)胞)的促增值作用,以增值細(xì)胞中的DNA合成或者酶活性作為指標(biāo),間接推算出細(xì)胞因子的生物學(xué)活性。 三、試劑與器材: 10%FCS-RPMI-1640培養(yǎng)液,刀豆蛋白A(ConA),3H-TdR,C57BL小鼠,IL-2標(biāo)準(zhǔn)品,PHA。 離心機(jī)、超凈臺(tái)、分光光度儀、細(xì)胞培養(yǎng)板等。 四、操作方法: 1.人外周血T細(xì)胞PBMC分離 2.PHA刺激產(chǎn)生IL-2 3.離心去沉淀回收上清 4.IL-2活性測(cè)定 5.CTLL-2細(xì)胞株培養(yǎng) 6.MTT摻入 7.測(cè)OD值570nm 8.結(jié)果分析 五、關(guān)鍵步驟與注意事項(xiàng): 1.CTLL-2細(xì)胞存活率應(yīng)大于95%。 2.因標(biāo)本中含IL-4等,可影響TL-2的測(cè)定,采用抗IL-4 mAb吸附劑出去IL-4。 3.整個(gè)操作過程中應(yīng)該注意無菌操作。 4.細(xì)胞培養(yǎng)時(shí)要做復(fù)孔并設(shè)陰、陽(yáng)性對(duì)照。 六、思考題: 1.誘導(dǎo)產(chǎn)生IL-2時(shí)需要注意哪些方面? 2.IL-4會(huì)對(duì)IL-2的測(cè)定產(chǎn)生影響,應(yīng)如何消除? 3.PHA誘導(dǎo)產(chǎn)生IL-2的機(jī)理是什么?

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