DNA异常甲基化在氡与香烟烟雾致BEAS-2B细胞恶性转化过程中的作用

发布时间：2018-01-05 17:04

本文关键词：DNA异常甲基化在氡与香烟烟雾致BEAS-2B细胞恶性转化过程中的作用　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:通过体外单独染氡、香烟及联合染毒诱导BEAS-2B细胞发生恶性转化,建立恶性转化细胞模型。通过检测细胞恶性转化过程中甲基化转移酶基因m RNA表达以及全基因组甲基化水平的改变,以及利用高通量甲基化芯片筛选单独及联合染毒发生DNA启动子区域异常甲基化的特异基因,探索氡、香烟联合致肺癌的机制,为氡、香烟联合致肺癌的防治提供理论依据。方法:将处于对数生长期的BEAS-2B细胞以1.5×105个接种于0.4μm Transwell膜中,细胞贴壁后将Transwell膜置于染毒腔内,毒气持续泵入在膜上方直接与细胞接触染毒,氧气浓度恒定于21%,水浴维持染毒腔温度在37℃,每次同时染毒3个。实验设对照组C、氡气染毒组Rn、香烟烟雾染毒组Sm和联合染毒组RS。使用CCK-8试剂盒检测不同氡、香烟染毒浓度后细胞存活率,确定合适的单独及联合染毒剂量。染氡两次后将Transwell膜中细胞消化,置于培养瓶中常规培养,待生长至对数生长期后进行下一代染氡,共染毒15代。对照组暴露于浓度为空气本底值的相同装置中,其余条件同染毒组。完成15代染毒并传代至20代后,流式细胞仪分析细胞周期、凋亡变化,软琼脂分析克隆形成,酶标仪检测全基因组甲基化水平,荧光定量PCR检测DNMT1、DNMT3A和DNMT3B基因m RNA表达情况。Illumina450k甲基化芯片筛选单独及联合染毒发生异常甲基化的特异基因,并检测SPDEF、CDK5RAP1、PTPRM和ABCG1基因m RNA表达情况。结果:(1)细胞恶性转化模型的建立。在染毒时间相同条件下,随染毒剂量升高,细胞存活率逐渐下降;氡、烟联合染毒CI值1,提示二者具有协同效应;确定氡染毒浓度和时间为20000Bq/m3,30min;香烟烟雾染毒浓度和时间分别为20%,10min。细胞周期显示染毒15代传代20代的细胞G1期明显缩短,S期明显延长,联合染毒组细胞S期延长最为明显,表明细胞处于增殖旺盛状态,其周期发生一定程度的失控,同时细胞凋亡率明显降低,呈现凋亡抑制状态。与对照组相比,染毒15代传代20代细胞软琼脂克隆形成能力已明显形成,联合染毒组细胞软琼脂克隆形成率已达到24.00±0.31(P0.05)。(2)全基因组甲基化水平及甲基化水平的改变:与对照组相比,单独及联合染毒组全基因组甲基化水平呈下降趋势,染毒15代传代20代细胞甲基化水平降至最低;染毒组细胞DNMT1基因表达明显降低,DNMT3A,DNMT3B基因表达明显升高,表明染毒后BEAS-2B细胞在发生了全基因组低甲基化的同时伴有部分CPG岛的高甲基化。(3)甲基化芯片筛选氡、烟单独及联合染毒特异性基因:利用Illumina450k甲基化芯片对Rn15-20,Rs15-20,Sm15-20进行检测,筛选出SPDEF,CDK5RAP1,PTPRM,ABCG1基因。联合染毒组异常甲基化基因CDK5RAP1,在细胞的周期与凋亡调控中占据重要地位;单独染氡组异常甲基化基因ABCG1,负责调控细胞内胆固醇的动态平衡;单独染烟组异常甲基化基因PTPRM,参与内皮细胞增殖负调节,内皮细胞迁移的负调控;氡、烟单独染毒组交集基因SPDEF,介导细胞增殖、分化和恶性变。对筛选基因m RNA表达情况进行进一步检测与分析,发现与对照组相比,联合染毒组CDK5RAP1 m RNA表达降低明显;单独染毒组SPDEF m RNA表达升高明显;氡染毒组ABCG1 m RNA表达升高明显;烟染毒组PTPRM m RNA表达降低明显(P0.05)。结论:1.建立了体外染氡、烟联合染毒诱发BEAS-2B细胞恶性转化模型,且氡、烟具有协同效应。2.恶性转化过程中,DNA甲基化转移酶m RNA表达发生改变,且全基因组甲基化水平降低。3.氡、烟单独及联合染毒发生异常甲基化的基因可能是单独及联合染毒致BEAS-2B细胞恶性转化的不同作用机制。
[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.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

【共引文献】