叶酸在MNNG致哈族DNMT1高表达细胞DNA损伤及相关信号通路中作用的研究

发布时间：2018-03-22 12:29

本文选题：食管癌　切入点：哈萨克族　出处：《新疆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:DNMT1高表达在DNA甲基化模式的转变和肿瘤的发生、发展中起促进作用。在前期已建立的哈萨克族食管上皮永生化细胞系的基础上,利用TALE技术构建DNMT1高表达的哈萨克族食管上皮细胞株,分析叶酸在MNNG致哈族食管上皮细胞DNMT1高表达细胞DNA损伤及相关信号通路调控机制中的作用,探讨叶酸对MNNG致细胞损伤过程中的干预作用,为食管癌预防和治疗提供理论依据。方法:将哈族食管上皮细胞及哈族食管上皮DNMT1高表达细胞分别分为三组,使用MNNG进行染毒并分别施以低浓度叶酸、中等浓度叶酸及高浓度叶酸干预,使用倒置荧光显微镜观察各组细胞生长情况;应用单细胞凝胶电泳实验(彗星实验)检测各组细胞DNA损伤情况;应用RT-PCR方法检测各组细胞PI3K-AKT通路中PP2A、PTEN、AKT基因mRNA表达水平;应用Western blot法检测各组细胞PI3K-AKT通路中PP2A、PTEN、AKT的蛋白表达水平。结果:(1)两种哈族食管上皮细胞形态损伤情况随染毒时间延长而加重,且在相同干预情况下,DNMT1高表达细胞较非高表达细胞受MNNG影响更为严重,但高浓度叶酸组细胞较同类型同时期低、中浓度叶酸组生长情况良好,细胞死亡率低,细胞排列整齐,形态正常,细胞镜下形态最接近于同类同时期对照组细胞;(2)两种食管上皮细胞DNA损伤情况随染毒时间延长而加重,且哈族食管上皮DNMT1高表达细胞较哈族食管上皮细胞DNA损伤情况更为严重,表现为尾长在染毒早期、中期、晚期时高于正常细胞组,差异有统计学意义(t早=2.043,P=0.004;t中=2.217,P=0.030;t晚=2.418,P=0.016),DNMT1高表达细胞组Olive尾矩长度在早期、中期及晚期时高于正常细胞组,差异有统计学意义(t早=2.471,P=0.020;t中=2.412,P=0.016;t晚=2.047,P=0.004)。但高浓度叶酸组细胞较同类型同时期低、中浓度叶酸组DNA损伤情况较轻,Tail DNA%含量、尾长、Olive尾矩均低于低、中浓度叶酸组(P0.05);(3)两种食管上皮细胞PP2A、PTEN基因mRNA表达水平及蛋白表达水平随染毒时间延长而降低,且DNMT1高表达细胞表达水平低于同时期同叶酸浓度哈族食管上皮细胞(P0.05),而AKT基因mRNA表达水平及蛋白表达水平随染毒时间延长而上升,且DNMT1高表达细胞AKT基因mRNA表达水平及蛋白表达水平高于同时期同叶酸浓度哈族食管上皮细胞(P0.05)。但高浓度叶酸组细胞较同类型同时期低、中浓度叶酸组PP2A、PTEN基因mRNA表达水平及蛋白表达水平较高,AKT基因mRNA表达水平及蛋白表达水平较低(P0.05)。结论:哈族DNMT1高表达细胞较哈族食管上皮细胞更易受到MNNG影响,且随染毒时间延长细胞损伤程度加重,表现为形态改变、DNA损伤,以及导致PI3K-AKT通路中PP2A、PTEN基因mRNA及蛋白表达下调,AKT基因mRNA及蛋白表达上调;但高浓度叶酸可降低MNNG对细胞的损伤,保护PI3K-AKT通路中PP2A、PTEN、AKT基因的正常表达,保持充足的叶酸摄入对食管癌的发生发展可起到一定的保护作用。
[Abstract]:Objective to promote the development of DNA methylation and tumorigenesis by overexpression of DNMT1 in Kazakh esophageal epithelial immortalized cell lines. A Kazakh esophageal epithelial cell line with high expression of DNMT1 was constructed by TALE technique. The role of folic acid in DNA damage and signal pathway regulation of DNMT1 overexpression cells induced by MNNG was analyzed. To investigate the effect of folic acid on the process of MNNG induced cell injury and to provide theoretical basis for the prevention and treatment of esophageal carcinoma. Methods: the esophageal epithelial cells of Kazakh nationality and the high expression cells of DNMT1 in esophageal epithelium of Kazakh nationality were divided into three groups respectively. Low concentration folic acid, middle concentration folic acid and high concentration folic acid were treated with MNNG, and the growth of each group was observed by inverted fluorescence microscope. Single cell gel electrophoresis (comet assay) was used to detect the DNA damage in each group, and RT-PCR method was used to detect the mRNA expression level of PP2An PTENK gene in the PI3K-AKT pathway of each group. Western blot method was used to detect the protein expression of PP2An PTENTENT-AKT in the PI3K-AKT pathway of each group. Results the morphological damage of esophageal epithelial cells of two kinds of Kazakh nationality was aggravated with the prolongation of exposure time. In the same intervention, the high expression cells of DNMT1 were more seriously affected by MNNG than those of non-high expression cells, but the cells of high concentration folic acid group were lower than those of the same type at the same time, the growth condition of middle concentration folic acid group was good, the cell death rate was low, and the cells were arranged neatly. The DNA damage of two kinds of esophageal epithelial cells was aggravated with the prolongation of the exposure time, and the morphology of the two kinds of esophageal epithelial cells was similar to that of the control cells at the same time. The DNA damage of esophageal epithelial cells in Kazak nationality was more serious than that in the esophageal epithelial cells of Kazak nationality. The length of tail was higher than that of normal cells in the early, middle and late stages of exposure. There was significant difference in the length of Olive tail moment in the early stage, middle stage and late stage of the high expression cell group (2.217P0.030t, 2.418). The length of Olive tail moment in the high expression cell group was higher than that in the normal cell group at the early, middle and late stages, and the difference was statistically significant in the early stage of 2.471P0.020t. The difference was 2.412P0.016t, 2.047P0.004t. However, the cell length of the high concentration folic acid group was lower than that of the normal cell group at the same time. In the middle concentration folic acid group, the DNA damage rate was lower than that in the middle concentration folic acid group, and the tail moment of Olive was lower than that in the middle concentration folic acid group. The mRNA expression level and the protein expression level of the PP2A PTEN gene in the esophageal epithelial cells decreased with the prolongation of the exposure time. The high expression level of DNMT1 was lower than that of P0.05 in esophageal epithelial cells of Kazak nationality at the same time, but the expression level of mRNA and protein of AKT gene increased with the prolongation of exposure time. The expression level of mRNA and protein of AKT gene in high expression cells of DNMT1 was higher than that in esophageal epithelial cells of Kazak nationality at the same concentration of folic acid at the same time, but the cells in the group of high concentration folic acid were lower than those in the same period. In folic acid group, the mRNA expression level and protein expression level of PP2An PTEN gene were higher than that of AK gene mRNA and protein expression level. Conclusion: the high expression of DNMT1 in Kazak nationality is more susceptible to the influence of MNNG than that of esophageal epithelial cells of Kazak nationality. With the prolongation of the time of exposure, the degree of cell damage was aggravated, which was characterized by morphological change and down-regulation of mRNA and protein expression in PI3K-AKT pathway, but high concentration of folic acid could reduce the damage caused by MNNG. To protect the normal expression of PP2A- PTENT-AKT gene in PI3K-AKT pathway and maintain sufficient folic acid intake may play a protective role in the occurrence and development of esophageal carcinoma.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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