DNA甲基化在氯乙烯致大鼠肝细胞遗传损伤中的作用

发布时间：2018-05-06 08:43

本文选题：氯乙烯 + 肝细胞　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:检测氯乙烯对大鼠DNA损伤作用,测定大鼠的全基因组DNA甲基化水平,检测癌基因KRAS,损伤修复基因CDKN2A、RASSF1A,DNA烷化损伤修复基因MGMT,抑癌基因SYK基因启动子区甲基化水平及mRNA的表达量改变,探讨氯乙烯致癌在遗传机制和表观遗传机制的关系。方法:选取96只健康大鼠,按体重随机分成4组,每组24只,分别为阴性对照组和低剂量(5mg/kg)、中剂量(25mg/kg)、高剂量(125mg/kg)三个氯乙烯染毒剂量组;腹腔注射,隔日染毒,每周三次。每组大鼠分别于6、8、12周随机处死8只,取其肝脏,应用彗星实验评价肝细胞DNA损伤水平,DNA甲基化定量检测试剂盒(比色法)检测大鼠肝细胞全基因组甲基化水平,选用甲基化特异性PCR(QMSP)和实时荧光定量PCR(QPCR)分别检测上述基因启动子区甲基化水平及mRNA的表达量。采用SPSS22.0对实验数据进行统计分析,本次实验数据符合正态或近似正态分布,采用单因素方差分析ANOVA进行组间比较,两两比较采用LSD法;相关性分析采用双变量相关分析,选用Pearson相关系数,显著性水平为α=0.05。结果:1、氯乙烯可诱发大鼠肝脏组织肝细胞坏死,肝脂肪变性,肝硬化等组织病理变化,大鼠肝细胞DNA损伤水平随着染毒剂量的增加和染毒时间的延长而升高,肝细胞全基因组甲基化水平在染毒组中明显均高于对照组,差异有统计学意义(P0.05)。2、大鼠肝细胞5种基因启动子区甲基化水平的改变:2.1各组别之间比较:氯乙烯染毒6周时,各染毒组大鼠肝细胞MGMT基因启动子区甲基化水平均高于对照组(P0.05),mRNA的表达量随着剂量的增加而升高;染毒8周时,染毒组KRAS、SYK甲基化水平随着染毒剂量的增加而下降,SYK mRNA的表达量随着染毒剂量的增加而升高;染毒12周时,RASSF1A、MGMT甲基化水平随着染毒剂量的增加而明显升高,RASSF1A mRNA表达量随着染毒剂量的增加而下降。2.2各染毒时间之间比较:对照组中,KRAS、CDKN2A、RASSF1A、MGMT、SYK基因启动子区甲基化水平及mRNA表达量在各染毒时间差异无统计学意义(P0.05);低剂量组中,MGMT、SYK甲基化水平随着染毒时间的延长而下降,MGMT mRNA表达量随着染毒时间的延长而升高;中剂量组中,KRAS、MGMT、SYK甲基化水平在8周和12周时低于6周(P0.05),mRNA表达量随着染毒时间的延长而升高;高剂量组中,CDKN2A、RASSF1A基因甲基化水平在6周和8周时低于12周(P0.05),RASSF1A mRNA表达量在6周和8周明显高于12周(P0.05)。3、大鼠肝细胞DNA损伤和相关基因甲基化相关性分析:大鼠肝细胞DNA损伤与RASSF1A、MGMT基因启动子区甲基化水平呈正相关关系(P0.05)。结论:1、氯乙烯可引起大鼠肝细胞DNA损伤增加,全基因组甲基化水平升高。2、氯乙烯可引起KRAS和SYK基因启动子区甲基化水平的下降,CDKN2A、MGMT甲基化水平的升高。在短期、低剂量下,氯乙烯可引起RASSF1A启动子区甲基化水平下降,mRNA表达量增加;但当染毒时间延长,染毒剂量增加时,RASS1A启动子区甲基化水平升高,mRNA表达量下降。3、氯乙烯引起RASSF1A、MGMT基因启动子区甲基化水平的升高可能影响大鼠肝细胞DNA损伤的修复,使大鼠肝细胞DNA损伤增加。
[Abstract]:Aim: to detect the effect of vinyl chloride on DNA injury in rats and to determine the level of DNA methylation in the whole genome of rats, detect the gene KRAS, damage repair gene CDKN2A, RASSF1A, DNA alkylation repair gene MGMT, the level of methylation and mRNA in the promoter region of the tumor suppressor gene SYK gene, and explore the genetic mechanism and apparent mechanism of the carcinogenesis of vinyl chloride. Methods: the relationship of genetic mechanism. Method: 96 healthy rats were randomly divided into 4 groups according to body weight, 24 rats in each group, which were negative control group and low dose (5mg/kg), middle dose (25mg/kg), high dose (125mg/kg) three chloroethylene exposure dose group; intraperitoneal injection, infected every week, three times per week. Rats in each group were killed at random for 8 rats in 6,8,12 weeks respectively. Liver cell DNA damage level was evaluated by comet assay. DNA methylation quantitative detection kit (colorimetric assay) was used to detect the whole genome methylation level of rat liver cells. Methylation specific PCR (QMSP) and real-time fluorescent quantitative PCR (QPCR) were used to detect the methylation level of the promoter region and the expression of mRNA respectively. SPSS22.0 The experimental data were statistically analyzed. The experimental data conformed to normal or approximately normal distribution. The single factor variance analysis ANOVA was used to compare group comparison, and 22 was compared with LSD; correlation analysis adopted bivariate correlation analysis and Pearson correlation coefficient was selected. The significant water level was alpha =0.05. results: 1, vinyl chloride could induce rat liver group. Hepatocyte necrosis, hepatic steatosis, liver cirrhosis and other histopathological changes, the level of DNA damage in hepatocytes of rats increased with the increase of dose and prolongation of time. The total genome methylation level of hepatocytes in the infected group was significantly higher than that in the control group. The difference has the significance of P0.05 (.2) and the initiation of 5 genes in rat liver cells. The changes in the level of methylation in the subregion: 2.1 the comparison between each group: the methylation water of the MGMT gene promoter region of the rat hepatocytes was higher than that of the control group (P0.05) at 6 weeks, and the expression of mRNA increased with the increase of the dose. At 8 weeks, the level of KRAS and SYK methylation decreased with the increase of the dose. The expression of SYK mRNA increased with the increase of the dose, and at 12 weeks, the level of RASSF1A and MGMT methylation increased obviously with the increase of the dose, and the RASSF1A mRNA expression decreased with the increase of the dose of the infected.2.2, and the comparison between the duration of the.2.2 exposure time: KRAS, CDKN2A, RASSF1A, MGMT, and the methylation of the SYK gene promoter region. There was no significant difference between the level and the expression of mRNA in the time of exposure. In the low dose group, the level of MGMT, SYK methylation decreased with the prolongation of the exposure time, and the expression of MGMT mRNA increased with the prolongation of the exposure time; in the middle dose group, the level of KRAS, MGMT and SYK methylation was lower than 6 weeks (P0.05) at 8 and 12 weeks, and the mRNA expression was associated with the low dose group. In the high dose group, the methylation level of CDKN2A and RASSF1A genes was lower than 12 weeks (P0.05) at 6 and 8 weeks in the high dose group. The RASSF1A mRNA expression was significantly higher than 12 weeks (P0.05).3 in the 6 and 8 weeks. The correlation of DNA damage and related gene methylation in rat hepatocytes was analyzed: DNA injury in rat liver cells and RASSF1A, MGMT gene initiation The methylation level in the subregion is positive correlation (P0.05). Conclusion: 1, ethylene chloride can cause the increase of DNA damage in rat hepatocytes and the increase of.2 in the whole genome methylation level. Chloroethylene can cause the decrease of methylation level in the promoter region of KRAS and SYK genes, the increase of CDKN2A, MGMT methylation level. In the short term and low dose, vinyl chloride can cause RASSF1A initiation. The methylation level of the promoter region decreased and the expression of mRNA increased, but when the time was prolonged and the dose increased, the level of methylation in the RASS1A promoter region increased, the expression of mRNA decreased by.3. The elevated methylation level of RASSF1A in the promoter region of the MGMT gene may affect the repair of DNA damage in the rat liver cells and the DNA loss of rat liver cells. The injury increased.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

【参考文献】