發(fā)布時(shí)間：2018-02-16 18:50

  本文關(guān)鍵詞： 鼻咽癌 Kank1基因 5-脫氧雜氮胞苷 去甲基化　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：本研究采用5-脫氧雜氮胞苷(5-Aza-CdR)對(duì)鼻咽癌不同細(xì)胞株進(jìn)行處理,檢測(cè)藥物對(duì)細(xì)胞Kank1基因mRNA和蛋白表達(dá)的影響；檢測(cè)Kank1基因啟動(dòng)子區(qū)甲基化變化情況；并分析腫瘤細(xì)胞生物學(xué)行為變化,為進(jìn)一步探討鼻咽癌的發(fā)生機(jī)制并尋找鼻咽癌治療的新方法,同時(shí)對(duì)去甲基化藥物5-脫氧雜氮胞苷在鼻咽癌中的應(yīng)用做初步探索,為5-脫氧雜氮胞苷在臨床中的應(yīng)用提供新思路。 方法：選用鼻咽癌細(xì)胞株5-8F,6-10B, CNE1, CNE2和正常鼻咽上皮細(xì)胞株NP69,再次驗(yàn)證Kank1基因在鼻咽癌細(xì)胞株中的低表達(dá),將相對(duì)最低表達(dá)的6-10B, CNE1細(xì)胞株設(shè)為不加任何處理的對(duì)照組和經(jīng)不同濃度5-Aza-CdR處理的實(shí)驗(yàn)組。利用實(shí)時(shí)熒光定量PCR及Western Blotting檢測(cè)用藥前后Kank1基因的表達(dá)變化；采用BSP克隆測(cè)序法檢測(cè)Kank1基因啟動(dòng)子區(qū)CpG島甲基化變化情況；同時(shí)檢測(cè)用藥前后細(xì)胞生物學(xué)行為變化,其中包括：相差顯微鏡觀察各實(shí)驗(yàn)組細(xì)胞形態(tài)學(xué)改變情況；采用MTT比色法檢測(cè)不同濃度5-Aza-CdR對(duì)細(xì)胞增殖的影響；利用流式細(xì)胞術(shù)檢測(cè)不同濃度5-Aza-CdR處理72h后的細(xì)胞凋亡率。 結(jié)果： 1.與正常鼻咽上皮細(xì)胞NP69相比,Kank1在鼻咽癌細(xì)胞株5-8F,6-10B, CNE1, CNE2中表達(dá)下調(diào)。 2.不同濃度5-Aza-CdR干預(yù)6-10B,CNE1細(xì)胞后其Kankl的表達(dá)得到不同程度上調(diào),其表達(dá)上調(diào)程度與藥物濃度成正比。 3. Western Blotting方法檢測(cè)經(jīng)10μM濃度5-Aza-CdR處理6-10B, CNE1細(xì)胞72h較未處理對(duì)照組其Kank1蛋白表達(dá)增強(qiáng)。 4.BSP克隆測(cè)序結(jié)果表明,經(jīng)藥物5-Aza-CdR處理的6-10B, CNE1細(xì)胞中Kank1基因啟動(dòng)子區(qū)域得到逆轉(zhuǎn),其平均甲基化百分?jǐn)?shù)分別為11.11%、13.33%,明顯低于未經(jīng)藥物處理組的甲基化化水平(分別為64.44%、64.44%,P0.01)。 5.MTT檢測(cè)發(fā)現(xiàn)5-Aza-CdR可以明顯抑制6-10B, CNE1細(xì)胞的生長與增殖。 6.流式細(xì)胞術(shù)檢測(cè)結(jié)果表明細(xì)胞經(jīng)藥物處理后其凋亡率明顯高于對(duì)照組,5-Aza-CdR能誘導(dǎo)細(xì)胞凋亡。 結(jié)論： 5-Aza-CdR能夠有效逆轉(zhuǎn)鼻咽癌細(xì)胞株6-10B, CNE1中Kank1基因的異常甲基化,恢復(fù)Kank1mRNA和蛋白的表達(dá),從而誘導(dǎo)腫瘤細(xì)胞凋亡,抑制鼻咽癌細(xì)胞生長。
[Abstract]:Objective: to investigate the effects of 5-deoxyazacytidine 5-Aza-CdR on the expression of Kank1 gene mRNA and protein in nasopharyngeal carcinoma (NPC) cells, and to detect the methylation of the promoter region of Kank1 gene. In order to explore the mechanism of nasopharyngeal carcinoma (NPC) and seek a new method for the treatment of nasopharyngeal carcinoma (NPC), the application of demethylated drug 5-deoxycytidine in nasopharyngeal carcinoma (NPC) was studied. To provide a new idea for the clinical application of 5-deoxyazacytidine. Methods: the low expression of Kank1 gene in nasopharyngeal carcinoma (NPC) cell lines 5-8FN6-10B, CNE1, CNE2 and NP69was confirmed again. The relative lowest expression of 6-10B, CNE1 cell line was divided into control group without any treatment and experimental group treated with 5-Aza-CdR at different concentrations. The expression of Kank1 gene was detected by real-time fluorescence quantitative PCR and Western Blotting. BSP clone sequencing method was used to detect the CpG island methylation in the promoter region of Kank1 gene, and the changes of cell biological behavior before and after treatment were also detected, including: phase contrast microscope was used to observe the changes of cell morphology in each experimental group. The effects of different concentrations of 5-Aza-CdR on cell proliferation were detected by MTT colorimetry, and the apoptotic rate of 5-Aza-CdR treated with different concentration of 5-Aza-CdR for 72 hours was detected by flow cytometry. Results:. 1.Compared with the normal nasopharyngeal epithelial cell line NP69, the expression of kank1 was down-regulated in the nasopharyngeal carcinoma cell lines 5-8FN6-10B, CNE1 and CNE2. 2. The expression of Kankl was up-regulated in different concentrations of 5-Aza-CdR, which was proportional to the drug concentration. 3. The expression of Kank1 protein in the CNE1 cells treated with 5-Aza-CdR at 10 渭 M for 72 h was higher than that in the untreated control group by Western Blotting assay. 4. The results of cloning and sequencing showed that the promoter region of Kank1 gene was reversed in 6-10Bcells treated with 5-Aza-CdR, and the average methylation percentage of Kank1 gene was 11.11and 13.33, respectively, which was significantly lower than that of untreated group (P 0.01). 5. MTT assay showed that 5-Aza-CdR could significantly inhibit the growth and proliferation of 6-10 B, CNE1 cells. 6. The results of flow cytometry showed that the apoptosis rate of the cells treated with drugs was significantly higher than that of the control group (5-Aza-CdR). Conclusion:. 5-Aza-CdR can effectively reverse the aberrant methylation of Kank1 gene and restore the expression of Kank1mRNA and protein in nasopharyngeal carcinoma cell line 6-10B, thus inducing apoptosis and inhibiting the growth of nasopharyngeal carcinoma cells.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.63

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