【摘要】：目的:1.構(gòu)建ARA55基因的真核表達載體并完成相關(guān)鑒定,研究ARA55基因過表達對CNE2鼻咽癌細胞生物學(xué)特性的影響;2.明確ARA55在TGFβ1介導(dǎo)的CNE2細胞上皮間質(zhì)轉(zhuǎn)化(EMT)及侵襲、遷移中的作用。方法:1.通過PCR擴增獲得ARA55基因全長cDNA序列,純化后用HindIII和XhoI雙酶切消化,經(jīng)T4DNA連接酶作用,連接至pCMV-C-EGFP真核表達載體,連接產(chǎn)物經(jīng)轉(zhuǎn)化、挑取克隆、擴增培養(yǎng)后,提取小量質(zhì)粒,進行DNA測序鑒定;用HindIII和XhoI內(nèi)切酶消化酶消化連接產(chǎn)物,瓊脂糖凝膠電泳鑒定。重組質(zhì)粒經(jīng)ZLip2000轉(zhuǎn)染至CNE2鼻咽癌細胞,熒光顯微鏡觀察EGFP的表達情況,免疫印跡檢測ARA55-EGFP融合蛋白的表達水平;通過CCK-8比色、劃痕修復(fù)實驗、Transwell小室、AnnexinV-PE/7-AAD雙熒光染色、DNA梯狀電泳及蛋白印跡檢測凋亡蛋白變化等實驗,探討ARA55過表達對CNE2鼻咽癌細胞生物學(xué)特性的影響。2.利用一定濃度的TGFβ1誘導(dǎo)CNE2細胞株中ARA55的表達,免疫印跡檢測ARA55蛋白及EMT相關(guān)標志物N-cadherin、Claudin-1等的表達變化;采用ARA55的siRNA質(zhì)粒,經(jīng)X-tremeGENEsiRNA轉(zhuǎn)染至CNE2細胞株;通過CCK-8比色、劃痕修復(fù)、Transwell侵襲遷移等實驗,研究TGFβ1介導(dǎo)的ARA55表達上調(diào)及沉默以ARA55表達對CNE2鼻咽癌細胞EMT及侵襲遷移的影響。結(jié)果:1.DNA測序及雙酶切分析顯示pCMV-ARA55-EGFP重組載體構(gòu)建成功。2.CCK-8比色、劃痕修復(fù)實驗、Transwell小室等結(jié)果顯示pCMV-ARA55-EGFP組細胞生長增殖、侵襲遷移能力明顯低于pCMV-C-EGFP空載體組和/或空白對照組(P0.05或0.01);AnnexinV-PE/7-AAD雙熒光染色及DNA梯狀電泳結(jié)果可見,pCMV-ARA55-EGFP組細胞產(chǎn)生凋亡,且凋亡率明顯高于pCMV-C-EGFP空載體組(P0.05);免疫印跡顯示pCMV-ARA55-EGFP組細胞Bcl-2表達下調(diào),CytochromeC表達上調(diào),同時伴有Caspase-9和Caspase-3的激活。3.TGFβ1誘導(dǎo)后,免疫印跡顯示ARA55在CNE2細胞中的表達上調(diào);ARA55誘導(dǎo)組細胞發(fā)生間質(zhì)樣改變,N-cadherin的表達上升,Claudin-1表達下降,同時細胞的生長增殖及侵襲遷移能力明顯高于對照組(P0.05或0.01);誘導(dǎo)表達的ARA55通過siRNA下調(diào)后,siRNA-ARA55組細胞生長增殖及侵襲遷移能力下降(P0.05或0.01)。結(jié)論:1.成功構(gòu)建pCMV-ARA55-EGFP重組載體;2.ARA55過表達可抑制CNE2鼻咽癌細胞生長增殖,誘導(dǎo)凋亡;3.ARA55參與了TGFβ1介導(dǎo)的CNE2細胞EMT及侵襲遷移過程。
[Abstract]:Objective: 1. The eukaryotic expression vector of ARA55 gene was constructed and identified to study the effect of overexpression of ARA55 gene on the biological characteristics of CNE2 nasopharyngeal carcinoma cells. 2. To investigate the role of ARA55 in TGF 尾 1 mediated epithelial stroma transformation, invasion and migration of CNE2 cells. Method: 1. The full-length cDNA sequence of ARA55 gene was obtained by PCR amplification, purified and digested by HindIII and XhoI, and ligated to pCMV-C-EGFP eukaryotic expression vector by T4DNA ligase. The ligated product was transformed, cloned and cultured. A small amount of plasmid was extracted and identified by DNA sequencing. The ligated products were digested by HindIII and XhoI endonuclease and identified by agarose gel electrophoresis. The recombinant plasmid was transformed into CNE2 nasopharyngeal carcinoma cells by ZLip2000. The expression of EGFP was observed by fluorescence microscope and the expression level of ARA55-EGFP fusion protein was detected by immunoblotting. The changes of apoptotic proteins were detected by CCK-8 colorimetric assay, scratch repair test, Transwell chamber, AnnexinV-PE/7-AAD double fluorescence staining, DNA ladder electrophoresis and Western imprinting. To investigate the effect of overexpression of ARA55 on the biological characteristics of CNE2 nasopharyngeal carcinoma cells. 2. The expression of ARA55 in CNE2 cell line was induced by a certain concentration of TGF 尾 1, the expression of ARA55 protein and EMT related marker N 鈮，

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