【摘要】：目的:1.構(gòu)建ARA55基因的真核表達(dá)載體并完成相關(guān)鑒定,研究ARA55基因過表達(dá)對(duì)CNE2鼻咽癌細(xì)胞生物學(xué)特性的影響;2.明確ARA55在TGFβ1介導(dǎo)的CNE2細(xì)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(kuò)增獲得ARA55基因全長(zhǎng)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(dá)載體,連接產(chǎn)物經(jīng)轉(zhuǎn)化、挑取克隆、擴(kuò)增培養(yǎng)后,提取小量質(zhì)粒,進(jìn)行DNA測(cè)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(xì)胞,熒光顯微鏡觀察EGFP的表達(dá)情況,免疫印跡檢測(cè)ARA55-EGFP融合蛋白的表達(dá)水平;通過CCK-8比色、劃痕修復(fù)實(shí)驗(yàn)、Transwell小室、AnnexinV-PE/7-AAD雙熒光染色、DNA梯狀電泳及蛋白印跡檢測(cè)凋亡蛋白變化等實(shí)驗(yàn),探討ARA55過表達(dá)對(duì)CNE2鼻咽癌細(xì)胞生物學(xué)特性的影響。2.利用一定濃度的TGFβ1誘導(dǎo)CNE2細(xì)胞株中ARA55的表達(dá),免疫印跡檢測(cè)ARA55蛋白及EMT相關(guān)標(biāo)志物N-cadherin、Claudin-1等的表達(dá)變化;采用ARA55的siRNA質(zhì)粒,經(jīng)X-tremeGENEsiRNA轉(zhuǎn)染至CNE2細(xì)胞株;通過CCK-8比色、劃痕修復(fù)、Transwell侵襲遷移等實(shí)驗(yàn),研究TGFβ1介導(dǎo)的ARA55表達(dá)上調(diào)及沉默以ARA55表達(dá)對(duì)CNE2鼻咽癌細(xì)胞EMT及侵襲遷移的影響。結(jié)果:1.DNA測(cè)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(shí)驗(yàn)、Transwell小室等結(jié)果顯示pCMV-ARA55-EGFP組細(xì)胞生長(zhǎng)增殖、侵襲遷移能力明顯低于pCMV-C-EGFP空載體組和/或空白對(duì)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(xì)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(xì)胞Bcl-2表達(dá)下調(diào),CytochromeC表達(dá)上調(diào),同時(shí)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(xì)胞中的表達(dá)上調(diào);ARA55誘導(dǎo)組細(xì)胞發(fā)生間質(zhì)樣改變,N-cadherin的表達(dá)上升,Claudin-1表達(dá)下降,同時(shí)細(xì)胞的生長(zhǎng)增殖及侵襲遷移能力明顯高于對(duì)照組(P0.05或0.01);誘導(dǎo)表達(dá)的ARA55通過siRNA下調(diào)后,siRNA-ARA55組細(xì)胞生長(zhǎng)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(dá)可抑制CNE2鼻咽癌細(xì)胞生長(zhǎng)增殖,誘導(dǎo)凋亡;3.ARA55參與了TGFβ1介導(dǎo)的CNE2細(xì)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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