【摘要】：目的：應(yīng)用RNAi干擾技術(shù)干擾肺癌A549細(xì)胞株中特異性AT序列結(jié)合蛋白-1(Special AT-rich sequence-binding protein-1，，SATB1)基因的表達(dá)，探討其對A549細(xì)胞凋亡的影響。方法：設(shè)計合成針對SATB1基因的shRNA表達(dá)質(zhì)粒(SATB1-shRNA)。采用陽離子脂質(zhì)體法將SATB1-shRNA轉(zhuǎn)染入肺癌A549細(xì)胞。分別采用RT-PCR、Western-Blot法檢測SATB1-shRNA轉(zhuǎn)染A549細(xì)胞中SATB1、Bcl2、BAX及Caspase-3的表達(dá)。FCM檢測SATB1-shRNA轉(zhuǎn)染的A549細(xì)胞凋亡率。結(jié)果：經(jīng)凝膠電泳、 DNA測序鑒定分析，成功構(gòu)建了SATB1-shRNA。 RT-PCR和Western-Blot結(jié)果顯示SATB1-shRNA轉(zhuǎn)染成功后可顯著下調(diào)A549細(xì)胞中SATB1mRNA和蛋白的表達(dá)水平（P0.05）。RT-PCR和Western-Blot結(jié)果顯示Bcl2表達(dá)水平下降（P0.05），SATB1-shRNA轉(zhuǎn)染組BAX、Caspase-3表達(dá)水平上調(diào)（P0.05）。FCM檢測結(jié)果示SATB1-shRNA轉(zhuǎn)染組細(xì)胞凋亡率顯著增加（P0.05）。結(jié)論：SATB1-shRNA經(jīng)轉(zhuǎn)染肺癌A549細(xì)胞能有效抑制細(xì)胞中SATB1mRNA和蛋白的表達(dá)水平，誘導(dǎo)細(xì)胞凋亡，其機(jī)制可能與下調(diào)Bcl2基因表達(dá)所引起的級聯(lián)效應(yīng)有關(guān)。
[Abstract]:Aim: to investigate the effect of RNAi interference on the expression of specific AT sequence binding protein-1 (Special AT-rich sequence-binding protein-1,SATB1) gene in A549 cell line. Methods: shRNA expression plasmid (SATB1-shRNA) targeting SATB1 gene was designed and synthesized. SATB1-shRNA was transfected into lung cancer A549 cells by cationic liposome method. RT-PCR,Western-Blot assay was used to detect the expression of SATB1,Bcl2,BAX and Caspase-3 in A549 cells transfected with SATB1-shRNA, and FCM was used to detect the apoptosis rate of A549 cells transfected with SATB1-shRNA. Results: SATB1-shRNA. was successfully constructed by gel electrophoresis and DNA sequencing. RT-PCR and Western-Blot showed that SATB1-shRNA transfection could significantly down-regulate the expression of SATB1mRNA and protein in A549 cells (P0.05). RT-PCR and Western-Blot showed a decrease in Bcl2 expression (P0.05), and SATB1-shRNA transfected BAX,. The expression of Caspase-3 was up-regulated (P0.05). FCM results showed that the apoptotic rate of SATB1-shRNA transfection group was significantly increased (P0.05). Conclusion: transfection of SATB1-shRNA into lung cancer A549 cells can effectively inhibit the expression of SATB1mRNA and protein and induce apoptosis. The mechanism may be related to the cascade effect caused by down-regulation of Bcl2 gene expression.
【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】：2012
【分類號】：R363

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相關(guān)期刊論文 前1條

1 周來勇;劉芳;童健;陳群請;張福偉;郭琳瑯;;實時熒光定量RT-PCR分析非小細(xì)胞肺癌SATB1的表達(dá)和臨床病理意義[J];南方醫(yī)科大學(xué)學(xué)報;2009年03期

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