本文關(guān)鍵詞：DNA異常甲基化在氡與香煙煙霧致BEAS-2B細(xì)胞惡性轉(zhuǎn)化過程中的作用　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 氡 煙 DNA甲基化 甲基化芯片 基因表達(dá)

【摘要】：目的:通過體外單獨(dú)染氡、香煙及聯(lián)合染毒誘導(dǎo)BEAS-2B細(xì)胞發(fā)生惡性轉(zhuǎn)化,建立惡性轉(zhuǎn)化細(xì)胞模型。通過檢測細(xì)胞惡性轉(zhuǎn)化過程中甲基化轉(zhuǎn)移酶基因m RNA表達(dá)以及全基因組甲基化水平的改變,以及利用高通量甲基化芯片篩選單獨(dú)及聯(lián)合染毒發(fā)生DNA啟動(dòng)子區(qū)域異常甲基化的特異基因,探索氡、香煙聯(lián)合致肺癌的機(jī)制,為氡、香煙聯(lián)合致肺癌的防治提供理論依據(jù)。方法:將處于對(duì)數(shù)生長期的BEAS-2B細(xì)胞以1.5×105個(gè)接種于0.4μm Transwell膜中,細(xì)胞貼壁后將Transwell膜置于染毒腔內(nèi),毒氣持續(xù)泵入在膜上方直接與細(xì)胞接觸染毒,氧氣濃度恒定于21%,水浴維持染毒腔溫度在37℃,每次同時(shí)染毒3個(gè)。實(shí)驗(yàn)設(shè)對(duì)照組C、氡氣染毒組Rn、香煙煙霧染毒組Sm和聯(lián)合染毒組RS。使用CCK-8試劑盒檢測不同氡、香煙染毒濃度后細(xì)胞存活率,確定合適的單獨(dú)及聯(lián)合染毒劑量。染氡兩次后將Transwell膜中細(xì)胞消化,置于培養(yǎng)瓶中常規(guī)培養(yǎng),待生長至對(duì)數(shù)生長期后進(jìn)行下一代染氡,共染毒15代。對(duì)照組暴露于濃度為空氣本底值的相同裝置中,其余條件同染毒組。完成15代染毒并傳代至20代后,流式細(xì)胞儀分析細(xì)胞周期、凋亡變化,軟瓊脂分析克隆形成,酶標(biāo)儀檢測全基因組甲基化水平,熒光定量PCR檢測DNMT1、DNMT3A和DNMT3B基因m RNA表達(dá)情況。Illumina450k甲基化芯片篩選單獨(dú)及聯(lián)合染毒發(fā)生異常甲基化的特異基因,并檢測SPDEF、CDK5RAP1、PTPRM和ABCG1基因m RNA表達(dá)情況。結(jié)果:(1)細(xì)胞惡性轉(zhuǎn)化模型的建立。在染毒時(shí)間相同條件下,隨染毒劑量升高,細(xì)胞存活率逐漸下降;氡、煙聯(lián)合染毒CI值1,提示二者具有協(xié)同效應(yīng);確定氡染毒濃度和時(shí)間為20000Bq/m3,30min;香煙煙霧染毒濃度和時(shí)間分別為20%,10min。細(xì)胞周期顯示染毒15代傳代20代的細(xì)胞G1期明顯縮短,S期明顯延長,聯(lián)合染毒組細(xì)胞S期延長最為明顯,表明細(xì)胞處于增殖旺盛狀態(tài),其周期發(fā)生一定程度的失控,同時(shí)細(xì)胞凋亡率明顯降低,呈現(xiàn)凋亡抑制狀態(tài)。與對(duì)照組相比,染毒15代傳代20代細(xì)胞軟瓊脂克隆形成能力已明顯形成,聯(lián)合染毒組細(xì)胞軟瓊脂克隆形成率已達(dá)到24.00±0.31(P0.05)。(2)全基因組甲基化水平及甲基化水平的改變:與對(duì)照組相比,單獨(dú)及聯(lián)合染毒組全基因組甲基化水平呈下降趨勢,染毒15代傳代20代細(xì)胞甲基化水平降至最低;染毒組細(xì)胞DNMT1基因表達(dá)明顯降低,DNMT3A,DNMT3B基因表達(dá)明顯升高,表明染毒后BEAS-2B細(xì)胞在發(fā)生了全基因組低甲基化的同時(shí)伴有部分CPG島的高甲基化。(3)甲基化芯片篩選氡、煙單獨(dú)及聯(lián)合染毒特異性基因:利用Illumina450k甲基化芯片對(duì)Rn15-20,Rs15-20,Sm15-20進(jìn)行檢測,篩選出SPDEF,CDK5RAP1,PTPRM,ABCG1基因。聯(lián)合染毒組異常甲基化基因CDK5RAP1,在細(xì)胞的周期與凋亡調(diào)控中占據(jù)重要地位;單獨(dú)染氡組異常甲基化基因ABCG1,負(fù)責(zé)調(diào)控細(xì)胞內(nèi)膽固醇的動(dòng)態(tài)平衡;單獨(dú)染煙組異常甲基化基因PTPRM,參與內(nèi)皮細(xì)胞增殖負(fù)調(diào)節(jié),內(nèi)皮細(xì)胞遷移的負(fù)調(diào)控;氡、煙單獨(dú)染毒組交集基因SPDEF,介導(dǎo)細(xì)胞增殖、分化和惡性變。對(duì)篩選基因m RNA表達(dá)情況進(jìn)行進(jìn)一步檢測與分析,發(fā)現(xiàn)與對(duì)照組相比,聯(lián)合染毒組CDK5RAP1 m RNA表達(dá)降低明顯;單獨(dú)染毒組SPDEF m RNA表達(dá)升高明顯;氡染毒組ABCG1 m RNA表達(dá)升高明顯;煙染毒組PTPRM m RNA表達(dá)降低明顯(P0.05)。結(jié)論:1.建立了體外染氡、煙聯(lián)合染毒誘發(fā)BEAS-2B細(xì)胞惡性轉(zhuǎn)化模型,且氡、煙具有協(xié)同效應(yīng)。2.惡性轉(zhuǎn)化過程中,DNA甲基化轉(zhuǎn)移酶m RNA表達(dá)發(fā)生改變,且全基因組甲基化水平降低。3.氡、煙單獨(dú)及聯(lián)合染毒發(fā)生異常甲基化的基因可能是單獨(dú)及聯(lián)合染毒致BEAS-2B細(xì)胞惡性轉(zhuǎn)化的不同作用機(jī)制。
[Abstract]:Objective: through in vitro alone radon, BEAS-2B cell malignant transformation induced by cigarette and joint exposure, establishment of malignant transformation cell model. Through the detection of cell malignant transformation of methyl transferase gene m RNA expression and change of genomic DNA methylation levels, as well as the use of high-throughput methylation microarray combined exposure of DNA promoter sub regional abnormal methylation of specific genes, explore the mechanism of radon induced lung cancer, cigarette radon, provide theoretical basis for the prevention of cigarette induced lung cancer. Methods: BEAS-2B cells in the exponential growth phase to 1.5 * 105 inoculation in 0.4 m Transwell film, adherent cells after Transwell film at in the cavity, gas pump in direct contact with the cells at the top of the membrane, the concentration of oxygen in water bath to maintain constant 21%, exposure cavity temperature at 37 degrees, each time exposure 3. Experimental design The control group C, radon exposure group Rn, cigarette smoke exposure group Sm and treatment group RS. using CCK-8 kit to detect the different radon exposure concentration after cigarette, cell survival rate, determine the combined exposure dose of radon. Right after two times of the Transwell cells in the digestive membrane, Zhi Yupei keep the bottle to be grown with conventional culture. To the logarithmic growth phase after the next generation of radon exposure, a total of 15 generations. The control group was exposed to the same concentration as the background value of the air device, other conditions being the same as exposure group. Complete the 15 generation exposure and the passage to the 20 generation, cell cycle analysis, flow cytometry apoptosis, soft agar cloning and analysis the formation, microplate DNA methylation, fluorescence quantitative PCR detection of DNMT1 gene specific.Illumina450k methylation microarray alone and combined with methylation of the DNMT3B gene expression of DNMT3A and m RNA, and the detection of SPDEF, CD K5RAP1, PTPRM and ABCG1 expression of M gene of RNA. Results: (1) to establish the model of malignant transformation of cells. In the exposure time under the same conditions, with the dose increased, the survival rate of cells decreased gradually; radon, smoke exposure combined with CI value of 1, indicating that the two has a synergistic effect; determining the radon concentration and time 20000Bq/m3,30min; cigarette smoke exposure concentration and time were 20%, 10min. cells exposed for 15 passages of the 20 generation of cells in G1 phase was significantly shortened, S was significantly prolonged, the combined group of cells in S phase increased the most obviously, that the proliferation of cells in the active state, the cycle control to a certain extent, at the same time, the rate of cell apoptosis showed significantly reduced, inhibition of apoptosis. Compared with the control group, after 15 passages cells of the 20 generation soft agar colony forming ability has obviously formed, soft agar clone formation rate of treatment group reached 24 + 0.31 (P0.0 5). (2) methylation and methyl genome-wide level changes: compared with the control group, the separate and combined group whole genome methylation level decreased, exposure to 15 passage cells of the 20 generation methylation level to the lowest; the expression of DNMT1 gene significantly decreased treated cells, DNMT3A, DNMT3B gene expression obviously, that after exposure of BEAS-2B cells in the genome-wide hypomethylation accompanied by partial CPG island hypermethylation. (3) methylation microarray radon, smoke separate and combined specific genes by Illumina450k methylation chip of Rn15-20, Rs15-20, Sm15-20 were detected and screened for SPDEF. CDK5RAP1, PTPRM, ABCG1 gene. The combined group of abnormal methylation of CDK5RAP1 gene, plays an important role in the regulation of cell cycle and apoptosis in the infected group alone; radon abnormal methylation of ABCG1 gene is responsible for the regulation of intracellular cholesterol. Dynamic balance; single cigarette exposure group of abnormal methylation of PTPRM gene in endothelial cell proliferation, negative regulation, negative regulation of endothelial cell migration; radon, smoke exposure group intersection gene SPDEF mediated cell proliferation, differentiation and malignant transformation. The expression for further detection and analysis of M RNA gene screening, compared with the control group, treatment group CDK5RAP1 m RNA expression decreased significantly; SPDEF exposure group M RNA expression increased significantly; expression of radon exposure group ABCG1 significantly increased m RNA expression; smoke exposure group PTPRM m RNA decreased significantly (P0.05). Conclusion: 1. the establishment of the in vitro combined exposure of radon, smoke induced BEAS-2B cell malignant transformation model and the smoke, radon, with the process of malignant transformation of synergistic effect of.2., DNA methyltransferase m RNA expression changes, and the whole genome methylation level decreased.3. alone and in combination with radon, smoke exposure methylation base It may be the different mechanism of the malignant transformation of BEAS-2B cells caused by single and combined poisoning.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 杜厚兵;香煙煙霧致BEAS-2B細(xì)胞惡性轉(zhuǎn)化中相關(guān)基因表達(dá)及啟動(dòng)子甲基化改變[D];蘇州大學(xué);2011年

2 劉玉萍;香煙煙霧致BEAS-2B細(xì)胞惡變過程中表觀遺傳改變研究[D];蘇州大學(xué);2014年

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本文編號(hào)：1384016