本文選題：食管癌　切入點：哈薩克族　出處：《新疆醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:DNMT1高表達在DNA甲基化模式的轉變和腫瘤的發(fā)生、發(fā)展中起促進作用。在前期已建立的哈薩克族食管上皮永生化細胞系的基礎上,利用TALE技術構建DNMT1高表達的哈薩克族食管上皮細胞株,分析葉酸在MNNG致哈族食管上皮細胞DNMT1高表達細胞DNA損傷及相關信號通路調控機制中的作用,探討葉酸對MNNG致細胞損傷過程中的干預作用,為食管癌預防和治療提供理論依據。方法:將哈族食管上皮細胞及哈族食管上皮DNMT1高表達細胞分別分為三組,使用MNNG進行染毒并分別施以低濃度葉酸、中等濃度葉酸及高濃度葉酸干預,使用倒置熒光顯微鏡觀察各組細胞生長情況;應用單細胞凝膠電泳實驗(彗星實驗)檢測各組細胞DNA損傷情況;應用RT-PCR方法檢測各組細胞PI3K-AKT通路中PP2A、PTEN、AKT基因mRNA表達水平;應用Western blot法檢測各組細胞PI3K-AKT通路中PP2A、PTEN、AKT的蛋白表達水平。結果:(1)兩種哈族食管上皮細胞形態(tài)損傷情況隨染毒時間延長而加重,且在相同干預情況下,DNMT1高表達細胞較非高表達細胞受MNNG影響更為嚴重,但高濃度葉酸組細胞較同類型同時期低、中濃度葉酸組生長情況良好,細胞死亡率低,細胞排列整齊,形態(tài)正常,細胞鏡下形態(tài)最接近于同類同時期對照組細胞;(2)兩種食管上皮細胞DNA損傷情況隨染毒時間延長而加重,且哈族食管上皮DNMT1高表達細胞較哈族食管上皮細胞DNA損傷情況更為嚴重,表現為尾長在染毒早期、中期、晚期時高于正常細胞組,差異有統計學意義(t早=2.043,P=0.004;t中=2.217,P=0.030;t晚=2.418,P=0.016),DNMT1高表達細胞組Olive尾矩長度在早期、中期及晚期時高于正常細胞組,差異有統計學意義(t早=2.471,P=0.020;t中=2.412,P=0.016;t晚=2.047,P=0.004)。但高濃度葉酸組細胞較同類型同時期低、中濃度葉酸組DNA損傷情況較輕,Tail DNA%含量、尾長、Olive尾矩均低于低、中濃度葉酸組(P0.05);(3)兩種食管上皮細胞PP2A、PTEN基因mRNA表達水平及蛋白表達水平隨染毒時間延長而降低,且DNMT1高表達細胞表達水平低于同時期同葉酸濃度哈族食管上皮細胞(P0.05),而AKT基因mRNA表達水平及蛋白表達水平隨染毒時間延長而上升,且DNMT1高表達細胞AKT基因mRNA表達水平及蛋白表達水平高于同時期同葉酸濃度哈族食管上皮細胞(P0.05)。但高濃度葉酸組細胞較同類型同時期低、中濃度葉酸組PP2A、PTEN基因mRNA表達水平及蛋白表達水平較高,AKT基因mRNA表達水平及蛋白表達水平較低(P0.05)。結論:哈族DNMT1高表達細胞較哈族食管上皮細胞更易受到MNNG影響,且隨染毒時間延長細胞損傷程度加重,表現為形態(tài)改變、DNA損傷,以及導致PI3K-AKT通路中PP2A、PTEN基因mRNA及蛋白表達下調,AKT基因mRNA及蛋白表達上調;但高濃度葉酸可降低MNNG對細胞的損傷,保護PI3K-AKT通路中PP2A、PTEN、AKT基因的正常表達,保持充足的葉酸攝入對食管癌的發(fā)生發(fā)展可起到一定的保護作用。
[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.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

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