苯致SD大鼠造血组织DNA损伤与XPD基因表达及甲基化的关系

发布时间：2018-06-02 12:50

本文选题：苯 + XPD基因　；参考：《贵州医科大学》2016年硕士论文

【摘要】：目的:探讨XPD基因甲基化及表达在苯致SD大鼠造血组织损伤中的作用及其可能的机制。方法:30只雄性SD大鼠被随机分为5组,每组6只,分别为空白对照组(未经任何处理)、溶剂对照组(玉米油)、苯低剂量组(200mg/kg)、苯中剂量组(400mg/kg)、苯高剂量组(800mg/kg),除空白对照组外,各处理组每天灌胃1次,连续28天。灌胃染毒结束后1.HE染色观察股骨的病理组织学结构改变;2.采用单细胞凝胶电泳(SCGE)检测外周血细胞及骨髓细胞DNA损伤情况;3.采用实时荧光定量PCR法检测骨髓细胞中XPD基因的mRNA转录水平;4.免疫印迹法(WB)检测骨髓细胞XPD蛋白的表达水平;5.采用重亚硫酸盐测序法(BSP)检测血液及骨髓细胞中XPD基因甲基化状态。结果:1.病理组织学结果显示,随着染毒剂量的增加,苯染毒组骨小梁出现明显断裂现象,软骨细胞层排列更加紊乱,破裂细胞增多,骨髓造血细胞减少,脂肪滴增多;2.SCGE结果提示,苯染毒可致外周血及骨髓细胞DNA损伤,且损伤效应随着染毒浓度增加而增大;3.Taqman探针实时定量PCR法结果显示,空白对照组、溶剂对照组、苯低、中、高剂量组骨髓细胞XPD mRNA相对表达量分别为0.600±0.0484、0.454±0.331、0.501±0.446、1.068±0.666、1.072±0.047,与空白对照组比较,苯各染毒组均表达升高,且苯染毒中、高剂量组与空白对照组比较差异有统计学意义(P0.05);4.WB检测结果显示,空白对照组、溶剂对照组、苯染毒低、中、高剂量组XPD蛋白的表达量分别为(102.78±2.32)%、(100.00±0.00)%、(135.68±13.67)%、(154.48±9.78)%、(176.55±11.05)%,苯染毒各剂量组与对照组比较,差异均有统计学意义(P0.05),且苯染毒各剂量组之间比较,差异具有统计学意义(P0.05);5.BSP结果显示,空白对照组、溶剂对照组、苯低、中、高剂量组的骨髓细胞甲基化率分别为0.60%、0.30%、1.40%、1.10%、0.80%,苯染毒组与对照组比较,差异无统计学意义(P0.05);各组外周血细胞甲基化率分别为2.20%、0.80%、3.10%、1.70%、2.20%,苯染毒各剂量组与对照组比较差异无统计学意义(P0.05)。结论:苯对SD大鼠血液及骨髓细胞均具有DNA损伤作用,其机制可能与上调XPD基因表达有关,而XPD基因甲基化可能未参与转录调控过程。
[Abstract]:Aim: to investigate the role and possible mechanism of methylation and expression of XPD gene in hematopoietic tissue injury of SD rats induced by benzene. Methods 30 male Sprague-Dawley rats were randomly divided into 5 groups, 6 rats in each group. They were divided into five groups: blank control group (without any treatment), solvent control group (corn oil, benzene low dose group, benzene low dose group), benzene middle dose group (400 mg / kg), benzene high dose group (800 mg / kg), except blank control group. Each treatment group was gavaged once a day for 28 days. The histopathological changes of femur were observed by 1.HE staining after oral administration. Single cell gel electrophoresis (SCGE) was used to detect DNA damage in peripheral blood cells and bone marrow cells. The mRNA transcription level of XPD gene in bone marrow cells was detected by real-time fluorescence quantitative PCR assay. The expression of XPD protein in bone marrow cells was detected by Western blot. The methylation status of XPD gene in blood and bone marrow cells was detected by bisulfite sequencing. The result is 1: 1. The histopathological results showed that the bone trabeculae in benzene exposed group were obviously broken with the increase of dose, the arrangement of chondrocytes was more disordered, the number of ruptured cells was increased, the hematopoietic cells of bone marrow were decreased, and the fat droplets were increased. Benzene exposure induced DNA damage in peripheral blood and bone marrow cells, and the damage effect increased with the increase of concentration of benzene. The results of real-time quantitative PCR with Taqman probe showed that: blank control group, solvent control group, benzene low, medium, middle, low, middle, low benzene, low benzene, The relative expression of XPD mRNA in bone marrow cells in the high dose group was 0.600 卤0.0484 卤0.454 卤0.331U 0.501 卤0.446U 1.068 卤0.666U 1.072 卤0.0477.Compared with the blank control group, the expression of XPD mRNA in the high dose group was higher than that in the blank control group, and the difference between the high dose group and the blank control group was statistically significant (P 0.05) 4.WB. The expression of XPD protein in the middle and high dose groups were 102.78 卤2.32, 100.00 卤0.005 卤135.68 卤13.67 and 154.48 卤9.780.55 卤11.05, respectively. The difference between each dose group of benzene exposure and the control group was statistically significant (P 0.05). The results of BSP showed that the methylation rates of bone marrow cells in the blank control group, solvent control group, benzene low, medium and high dose group were 0.600.30 and 1.40 and 1.100.80, respectively. The methylation rate of peripheral blood cells in each group was 2.20 / 0.80 and 3.100.70 / 2.20 respectively. There was no significant difference between each dose group and the control group (P 0.05). Conclusion: benzene can damage DNA in blood and bone marrow cells of SD rats, and its mechanism may be related to up-regulation of XPD gene expression, while methylation of XPD gene may not participate in the process of transcriptional regulation.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R114

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