芳香烃受体及肝脏代谢酶在饮用水有机提取物染毒大鼠肝脏中的表达及意义

发布时间：2018-07-23 20:55

【摘要】：目的:利用课题组前期制备的饮用水有机提取物致大鼠肝损伤动物模型,对芳香烃受体及下游调控基因(肝脏代谢酶)的表达及活性进行观察,探讨饮用水有机污染物的异常肝脏生物转化在肝损伤中的作用。方法:于2012年7-9月(丰水期)采集该地的末梢水水样并制备有机提取物。将50只清洁级SD大鼠随机分为5组,分别为空白对照组、溶剂对照(玉米油)组和饮用水有机提取物低中高3个染毒组(剂量分别为5L/kg、20L/kg、80L/kg),每组10只,雌雄各半。采用经口灌胃方式进行染毒,每天1次,连续染毒12周。制备大鼠血清,采用分光光度法检测天冬氨酸转氨酶(Aspartate aminotransferas,AST)、丙氨酸转氨酶(Alanine amunotransferase,ALT)、胆碱酯酶(Cholinesterase,CHE)的活力及总蛋白(Total protein,TP)、白蛋白(Albumin,ALB)的含量;提取肝组织总RNA,采用实时荧光定量PCR(RT-q RCR)法检测芳香烃受体(aryl hydrocarbon receptor,Ah R)、热休克蛋白90(heat shock protein 90,HSP90)、芳香烃受体核转位蛋白(aryl hydrocarbon nuclear translocator,ARNT)、细胞色素1A2(CYP1A2)、细胞色素2E1(CYP2E1)、谷胱甘肽-S-转移酶A1(glutathione-S-transferase A1,GSTA1)的m RNA表达情况;提取肝组织总蛋白,采用蛋白印记(Western Blot)法检测Ah R、HSP90、ARNT、CYP1A2、CYP2E1、GSTA1的蛋白表达情况;制备肝匀浆,采用分光光度法检测谷胱甘肽-S-转移酶(glutathione-S-trans ferase,GSTs)活力;制备肝微粒体,荧光分光光度法和活性比色法分别测定CYP1A2与CYP2E1酶活性;采用Spearman相关对芳香烃受体和肝脏代谢酶及肝损伤的相关性进行分析。结果:(1)随着染毒剂量的增加,大鼠血清CHE活力在中、高剂量组明显升高,而ALT、AST活力仅在高剂量组升高(P0.05);与对照组相比,TP和ALB水平在高剂量组明显下降有统计学意义(P0.05)。(2)与对照组相比,HSP90的m RNA和蛋白表达水平在低、中、高剂量组都明显升高,而Ah R与ARNT的m RNA和蛋白表达水平仅在中、高剂量组升高(P0.05)。(3)与对照组相比,CYP1A2与GSTA1的m RNA和蛋白表达水平在中、高剂量组明显升高,而CYP2E1的m RNA和蛋白表达水平仅在高剂量组升高(P0.05);随着染毒剂量的增加,高剂量组GSTA1 m RNA和蛋白表达水平比中剂量组有所降低,差异具有统计学意义(P0.05);CYP1A2和GSTs酶活性在中剂量与高剂量组显著增加,而CYP2E1酶活性仅在高剂量组增加(P0.05);与中剂量组相比,高剂量组GSTs的酶活性则明显降低(P0.05)。(4)肝脏代谢酶CYP1A2、GSTs酶活性与Ah R蛋白表达水平均呈正相关关系;染毒大鼠血清CHE水平与Ah R的蛋白表达水平及CYP1A2、CYP2E1、GSTs酶活性均呈正相关关系。结论:(1)在本实验条件下,较高剂量饮用水有机提取物暴露可活化芳香烃受体Ah R,从而诱导其配体HSP90及ARNT的转录表达和翻译表达。(2)芳香烃受体通路的异常上调,调控下游基因代谢酶CYP1A2与GSTA1的m RNA和蛋白表达增强,诱导代谢酶活性升高。(3)肝脏代谢酶活性异常升高,在对饮用水有机污染物解毒代谢的过程中,诱导自由基和毒性中间代谢物的产生,破坏细胞结构,造成肝细胞损伤。
[Abstract]:Objective: To observe the expression and activity of aromatic hydrocarbon receptor and downstream regulatory gene (liver metabolic enzyme), and to explore the role of abnormal liver biotransformation of drinking water organic pollutants in the liver damage caused by the organic extracts of drinking water prepared by the group in the previous period. Methods: in 7-9 months of 2012 (Fengshui period) 50 clean SD rats were randomly divided into 5 groups: blank control group, solvent control (corn oil) group and drinking water organic extract in 3 low middle and high toxic groups (dose of 5L/kg, 20L/kg, 80L/kg), each group of 10 rats and male and male. 1 times, the rat serum was prepared for 12 weeks. Aspartate aminotransferas (AST), alanine aminotransferase (Alanine amunotransferase, ALT), the activity of cholinesterase (Cholinesterase, CHE), the total protein (Total protein, TP), the content of albumin (Albumin,) were extracted by spectrophotometric method. The detection of aromatic hydrocarbon receptor (aryl hydrocarbon receptor, Ah R), heat shock protein 90 (heat shock protein 90, HSP90), aromatic hydrocarbon receptor nuclear transposition protein, cytochrome aryl, glutathione transferase (Ah) were detected by real time fluorescence quantitative PCR (RCR) method. The expression of M RNA in one-S-transferase A1, GSTA1, the total protein of liver tissue was extracted, and the protein expression of Ah R, HSP90, ARNT, CYP1A2, and Western Blot method was used to detect the protein expression of Ah, and the liver homogenate was prepared by spectrophotometric method to detect the activity of glutathione transferase, and the preparation of liver microsomes was made. The activity of CYP1A2 and CYP2E1 enzyme were measured by fluorescence spectrophotometry and activity colorimetry, and the correlation of Spearman correlation to the metabolic enzymes of liver and liver was analyzed by Spearman correlation. Results: (1) with the increase of dose, the activity of CHE in the serum of rats increased significantly, and the activity of ALT and AST was only in high dose group. Higher (P0.05). Compared with the control group, the level of TP and ALB decreased significantly in the high dose group (P0.05). (2) compared with the control group, the m RNA and protein expression level of HSP90 increased significantly in low, middle and high dose groups, while Ah R and ARNT m RNA and protein expression levels were only in the middle, high dose group increased. (3) compared with the control group, the level of Ah R and ARNT increased. With the level of M RNA and protein expression of GSTA1, the high dose group increased obviously, while the m RNA and protein expression level of CYP2E1 increased only in the high dose group (P0.05). With the increase of the dose, the GSTA1 m RNA and protein expression level in the high dose group was lower than that in the middle dose group, and the difference was statistically significant (P0.05); CYP1A2 and enzyme activity were in the high dose group. The medium dose and high dose group increased significantly, while the activity of CYP2E1 enzyme increased only in the high dose group (P0.05). Compared with the medium dose group, the activity of GSTs in the high dose group decreased significantly (P0.05). (4) the liver metabolic enzyme CYP1A2, the activity of GSTs enzyme and the expression level of Ah R protein were all Cheng Zhengxiang; the serum CHE level and Ah R protein expression water in the rats were infected. The activities of CYP1A2, CYP2E1 and GSTs enzyme were positively correlated. (1) under this experimental condition, the exposure of organic extracts from high doses of drinking water can activate the aromatic hydrocarbon receptor Ah R, thus inducing the transcription and expression of the ligand HSP90 and ARNT. (2) the abnormal up regulation of the aromatic hydrocarbon receptor pathway and the regulation of the downstream gene metabolic enzyme CYP1A2 and G The expression of M RNA and protein in STA1 is enhanced, and the activity of induced metabolic enzyme is increased. (3) the activity of metabolic enzymes in the liver is abnormal. It induces the production of free radicals and toxic intermediate metabolites in the process of detoxification and metabolism of organic pollutants in drinking water, destroys the structure of cells and causes damage to the liver cells.
【学位授予单位】：贵阳医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114

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