铅暴露环境下干预HO-1调控HMGB1表达的研究
发布时间：2018-08-16 10:44
【摘要】：本论文研究了铅暴露环境下HMGB1和HO-1表达的变化,以及干预HO-1对铅暴露环境中HMGB1表达的影响。第一部分铅暴露对断乳仔鼠海马组织HMGB1和HO-1表达的影响目的建立铅暴露断乳仔鼠模型,检测断乳仔鼠血液和海马组织中的铅含量,以及断乳仔鼠海马组织中HMGB1和HO-1的表达。方法1、动物建模:在检测到雌鼠受孕当天,将其随机分成3个组:(1)C组:空白对照组,给予双蒸水;(2)L组:低剂量铅暴露组,给予0.05%(0.5 g/L)醋酸铅水溶液;(3)H组:高剂量铅暴露组,给予0.2%(2.0 g/L)醋酸铅溶液。采用自由饮水方式进行铅暴露,直至仔鼠断乳(出生后21d)。2、实验方法:(1)采用Morris水迷宫对断乳仔鼠进行行为学测试;(2)采用电感耦合等离子体发射光谱仪(Inductively coupled plasma-atomic emission spectroscopy,ICP-AES)检测断乳仔鼠全血和海马组织中铅含量;(3)采用逆转录-聚合酶链反应(Reverse transcriptase polymerase chain reaction,RT-PCR)检测断乳仔鼠海马组织HMGB1和HO-1 mRNA水平;(4)采用蛋白免疫印迹法(Western blot,WB)检测断乳仔鼠海马组织HMGB1和HO-1蛋白水平;(5)采用免疫荧光法(Immunofluorescent,IF)检测HMGB1和HO-1在断乳仔鼠在海马组织各区域的蛋白水平。结果1、Mirros水迷宫实验结果:铅暴露会导致断乳仔鼠的学习记忆能力下降。(1)逃避潜伏期:与C组比较,L组和H组的潜伏期时间明显延长(P0.05);(2)穿越平台次数:与C组比较,L组和H组的穿越平台次数明显减少(P0.05),且H组的次数明显低于L组(P0.05)。2、血铅和海马组织铅含量的检测结果:铅暴露会引起断乳仔鼠的血铅和海马铅水平的上升。ICP-AES检测表明,断乳仔鼠血铅浓度和海马铅含量均随着铅暴露浓度的升高明显增高(P0.05)。3、铅暴露对断乳仔鼠HMGB1表达的影响:(1)RT-PCR实验发现,断乳仔鼠海马组织HMGB1 mRNA水平,会随着铅暴露浓度的升高明显增高(P0.05);(2)WB实验发现,HMGB1水平,随着铅暴露浓度的升高明显增高(P0.05);(3)IF实验发现,海马组织CA1、CA3以及DG区HMGB1蛋白水平,随着铅暴露浓度的升高明显增高(P0.05)。4、铅暴露对断乳仔鼠HO-1表达的影响:(1)RT-PCR实验发现,断乳仔鼠海马组织HO-1 mRNA水平,会随着铅暴露浓度的升高明显增高(P0.05);(2)WB实验发现,断乳仔鼠HO-1蛋白水平,随着铅暴露浓度的升高明显增高(P0.05);(3)IF实验发现,海马组织CA1、CA3以及DG区HO-1蛋白水平,随着铅暴露浓度的升高明显增高(P0.05)。第二部分铅暴露对PC12细胞HMGB1和HO-1表达的影响目的建立铅暴露PC12细胞模型,检测HMGB1和HO-1的表达。方法1、细胞建模:将PC12细胞分为3组:0μM(空白对照组)、1μM和100μM的醋酸铅,进行染铅处理24 h。2、实验方法:(1)采用RT-PCR检测HMGB1和HO-1在PC12细胞的mRNA水平;(2)采用WB检测HMGB1和HO-1在PC12细胞的蛋白水平;(3)采用IF检测铅暴露不同时间段(24h、48h、72h)HMGB1和HO-1在PC12细胞的蛋白水平。结果1、铅暴露对PC12细胞HMGB1表达的影响:(1)RT-PCR实验发现,HMGB1mRNA水平明显增高,与铅暴露浓度呈浓度依赖性关系(P0.05);(2)WB实验发现:HMGB1蛋白水平明显增高,与铅暴露浓度呈浓度依赖性关系(P0.05);(3)IF实验发现,铅暴露不同时间(24h、48h、72h)PC12细胞HMGB1蛋白水平明显增高,与铅暴露浓度呈浓度依赖性关系(P0.05)。2、铅暴露对PC12细胞HO-1表达的影响:(1)RT-PCR实验发现,HO-1 mRNA水平明显增高,与铅暴露浓度呈浓度依赖性关系(P0.05);(2)WB实验发现:HO-1蛋白水平明显增高,与铅暴露浓度呈浓度依赖性关系(P0.05);(3)IF实验发现,铅暴露不同时间(24h、48h、72h)PC12细胞HO-1蛋白水平明显增高,与铅暴露浓度呈浓度依赖性关系(P0.05)。第三部分铅暴露环境下干预HO-1的表达对HMGB1表达的影响目的在铅暴露细胞模型中加入HO-1的抑制剂ZnPP-Ⅸ或激动剂CoPP,观察HMGB1表达的变化。方法1、细胞建模:将PC12细胞分为4组:(1)S组:空白对照组,0μM PbAc;(2)PA组:PbAc(1μM);(3)Zn组:PbAc(1μM)+ZnPP(20μM)和(4)Co组:PbAc(1μM)+CoPP(20μM)。铅暴露处理12h时再加入干扰剂处理12h。2、实验方法:(1)采用RT-PCR检测HO-1和HMGB1在PC12细胞的mRNA水平;(2)采用WB检测HO-1和HMGB1在PC12细胞的蛋白水平。结果1、(1)与S组比,PA组和Co组HO-1 mRNA水平和蛋白水平明显增高(P0.05);(2)与PA组相比,Zn组HO-1表达明显下降(P0.05),Co组HO-1表达明显增高(P0.05)。2、(1)与S组比,PA组和Zn组HMGB1 mRNA水平和蛋白水平明显增高(P0.05);(2)与PA组相比,Zn组HMGB1表达明显增高(P0.05),Co组HMGB1表达明显下降(P0.05)。结论:1、铅暴露可以上调HMGB1和HO-1的表达。2、上调HO-1可以抑制铅暴露环境下HMGB1的表达。
[Abstract]:In this paper, we studied the changes of HMGB1 and HO-1 expression in lead-exposed environment, and the effect of HO-1 intervention on HMGB1 expression in lead-exposed environment. Part I Effects of lead exposure on HMGB1 and HO-1 expression in hippocampus of weaned offspring. Objective To establish a lead-exposed weaned offspring model, detect the lead content in blood and hippocampus, and Methods 1. Animal modeling: On the day of gestation, female rats were randomly divided into three groups: (1) C group: blank control group, given double-steamed water; (2) L group: low-dose lead exposure group, given 0.05% (0.5 g/L) lead acetate solution; (3) H group: high-dose lead exposure group, given 0.2% (2.0 g/L) lead acetate solution. (2) Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to detect the whole blood and hippocampus of weaned offspring. (3) Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the levels of HMGB1 and HO-1 mRNA in the hippocampus of weaned offspring; (4) Western blot (WB) was used to detect the levels of HMGB1 and HO-1 protein in the hippocampus of weaned offspring; (5) Immunofluorescence assay (Immuno) Fluorescent, IF) was used to detect the protein levels of HMGB1 and HO-1 in the hippocampus of weaned offspring. Results 1. Mirros water maze experiment showed that lead exposure could lead to the decline of learning and memory ability of weaned offspring. (1) Evasive latency: Compared with group C, the latency of group L and group H was significantly prolonged (P 0.05); (2) The number of crossing platforms: compared with group C. The number of plateau crossing in group L and group H was significantly decreased (P 0.05), and the number of plateau crossing in group H was significantly lower than that in group L (P 0.05). The results of blood lead and hippocampal lead content detection showed that lead exposure could cause the increase of blood lead and hippocampal lead levels in weaned offspring. The expression of HMGB1 in the hippocampus of weaned offspring was significantly increased with the increase of lead exposure (P 0.05). The levels of HMGB1 protein in CA1, CA3 and DG regions increased significantly with the increase of lead exposure concentration (P 0.05). 4. The effect of lead exposure on the expression of HO-1 in the hippocampus of weaned offspring rats: (1) RT-PCR showed that the level of HO-1 mRNA in the hippocampus of weaned offspring rats increased significantly with the increase of lead exposure concentration (P 0.05); (2) WB showed that the level of HO-1 protein in weaned offspring rats increased with the increase of lead exposure concentration (P 0.05). (3) IF assay showed that the levels of CA1, CA3 and HO-1 protein in hippocampus increased significantly with the increase of lead exposure (P 0.05). (2) Effects of lead exposure on the expression of HMGB1 and HO-1 in PC12 cells Objective To establish a lead-exposed PC12 cell model and detect the expression of HMGB1 and HO-1. Cell modeling: PC12 cells were divided into three groups: 0 mu M (blank control group), 1 mu M and 100 mu M lead acetate, and treated with lead for 24 h.2. Methods: (1) The mRNA levels of HMGB1 and HO-1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HMGB1 and HO-1 in PC12 cells were detected by WB; (3) IF was used to detect HMGB in different lead exposure periods (24h, 48h, 72h). Results (1) The expression of HMGB1 in PC12 cells was significantly increased by lead exposure. (1) The expression of HMGB1 mRNA was significantly increased by RT-PCR, which was in a concentration-dependent relationship with lead exposure (P 0.05); (2) The level of HMGB1 protein was significantly increased by WB, and was in a concentration-dependent relationship with lead exposure (P 0.05); (3) IF test. It was found that the HMGB1 protein level in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and was in a concentration-dependent relationship with lead exposure (P 0.05). The level of HO-1 protein in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and showed a concentration-dependent relationship with lead exposure concentration (P 0.05). The third part was the effect of intervention of HO-1 expression on HMGB1 expression in lead exposure environment. Methods 1. Cell modeling: PC12 cells were divided into 4 groups: (1) S group: blank control group, 0 mu M PbAc; (2) PA group: PbAc (1 mu M); and (3) Zn group: PbAc (1 mu M) + ZnPP (20 mu M) and (4) Co group: PbAc (1 mu M) + CoPP (20 mu) after 12 hours of lead exposure. Methods: (1) The mRNA levels of HO-1 and HMGB1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HO-1 and HMGB1 in PC12 cells were detected by WB. Results (1) Compared with S group, HO-1 mRNA and protein levels in PA group and Co group were significantly increased (P 0.05); (2) Compared with PA group, HO-1 expression in Zn group was significantly decreased (P 0.05), and HO-1 expression in Co group was significantly increased (P 0.05). HMGB1 mRNA and protein levels in PA group and Zn group were significantly higher than those in S group (P 0.05). (2) Compared with PA group, HMGB1 expression in Zn group was significantly higher (P 0.05) and HMGB1 expression in Co group was significantly lower (P 0.05). Conclusion: 1. Lead exposure can up-regulate the expression of HMGB1 and HO-1.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2185748
[Abstract]:In this paper, we studied the changes of HMGB1 and HO-1 expression in lead-exposed environment, and the effect of HO-1 intervention on HMGB1 expression in lead-exposed environment. Part I Effects of lead exposure on HMGB1 and HO-1 expression in hippocampus of weaned offspring. Objective To establish a lead-exposed weaned offspring model, detect the lead content in blood and hippocampus, and Methods 1. Animal modeling: On the day of gestation, female rats were randomly divided into three groups: (1) C group: blank control group, given double-steamed water; (2) L group: low-dose lead exposure group, given 0.05% (0.5 g/L) lead acetate solution; (3) H group: high-dose lead exposure group, given 0.2% (2.0 g/L) lead acetate solution. (2) Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to detect the whole blood and hippocampus of weaned offspring. (3) Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the levels of HMGB1 and HO-1 mRNA in the hippocampus of weaned offspring; (4) Western blot (WB) was used to detect the levels of HMGB1 and HO-1 protein in the hippocampus of weaned offspring; (5) Immunofluorescence assay (Immuno) Fluorescent, IF) was used to detect the protein levels of HMGB1 and HO-1 in the hippocampus of weaned offspring. Results 1. Mirros water maze experiment showed that lead exposure could lead to the decline of learning and memory ability of weaned offspring. (1) Evasive latency: Compared with group C, the latency of group L and group H was significantly prolonged (P 0.05); (2) The number of crossing platforms: compared with group C. The number of plateau crossing in group L and group H was significantly decreased (P 0.05), and the number of plateau crossing in group H was significantly lower than that in group L (P 0.05). The results of blood lead and hippocampal lead content detection showed that lead exposure could cause the increase of blood lead and hippocampal lead levels in weaned offspring. The expression of HMGB1 in the hippocampus of weaned offspring was significantly increased with the increase of lead exposure (P 0.05). The levels of HMGB1 protein in CA1, CA3 and DG regions increased significantly with the increase of lead exposure concentration (P 0.05). 4. The effect of lead exposure on the expression of HO-1 in the hippocampus of weaned offspring rats: (1) RT-PCR showed that the level of HO-1 mRNA in the hippocampus of weaned offspring rats increased significantly with the increase of lead exposure concentration (P 0.05); (2) WB showed that the level of HO-1 protein in weaned offspring rats increased with the increase of lead exposure concentration (P 0.05). (3) IF assay showed that the levels of CA1, CA3 and HO-1 protein in hippocampus increased significantly with the increase of lead exposure (P 0.05). (2) Effects of lead exposure on the expression of HMGB1 and HO-1 in PC12 cells Objective To establish a lead-exposed PC12 cell model and detect the expression of HMGB1 and HO-1. Cell modeling: PC12 cells were divided into three groups: 0 mu M (blank control group), 1 mu M and 100 mu M lead acetate, and treated with lead for 24 h.2. Methods: (1) The mRNA levels of HMGB1 and HO-1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HMGB1 and HO-1 in PC12 cells were detected by WB; (3) IF was used to detect HMGB in different lead exposure periods (24h, 48h, 72h). Results (1) The expression of HMGB1 in PC12 cells was significantly increased by lead exposure. (1) The expression of HMGB1 mRNA was significantly increased by RT-PCR, which was in a concentration-dependent relationship with lead exposure (P 0.05); (2) The level of HMGB1 protein was significantly increased by WB, and was in a concentration-dependent relationship with lead exposure (P 0.05); (3) IF test. It was found that the HMGB1 protein level in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and was in a concentration-dependent relationship with lead exposure (P 0.05). The level of HO-1 protein in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and showed a concentration-dependent relationship with lead exposure concentration (P 0.05). The third part was the effect of intervention of HO-1 expression on HMGB1 expression in lead exposure environment. Methods 1. Cell modeling: PC12 cells were divided into 4 groups: (1) S group: blank control group, 0 mu M PbAc; (2) PA group: PbAc (1 mu M); and (3) Zn group: PbAc (1 mu M) + ZnPP (20 mu M) and (4) Co group: PbAc (1 mu M) + CoPP (20 mu) after 12 hours of lead exposure. Methods: (1) The mRNA levels of HO-1 and HMGB1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HO-1 and HMGB1 in PC12 cells were detected by WB. Results (1) Compared with S group, HO-1 mRNA and protein levels in PA group and Co group were significantly increased (P 0.05); (2) Compared with PA group, HO-1 expression in Zn group was significantly decreased (P 0.05), and HO-1 expression in Co group was significantly increased (P 0.05). HMGB1 mRNA and protein levels in PA group and Zn group were significantly higher than those in S group (P 0.05). (2) Compared with PA group, HMGB1 expression in Zn group was significantly higher (P 0.05) and HMGB1 expression in Co group was significantly lower (P 0.05). Conclusion: 1. Lead exposure can up-regulate the expression of HMGB1 and HO-1.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
【参考文献】
相关期刊论文 前1条
1 俞静;吴国华;林清;周仁华;陆冬青;王佳;刘乾;冯秀艳;;HO-1激动剂Copp对放射所致内皮细胞凋亡的抑制作用[J];上海交通大学学报(医学版);2007年12期
相关硕士学位论文 前1条
1 叶伟峰;铅暴露大鼠脑区钙离子/钙调素关联信号通路及药物调控研究[D];浙江大学;2012年
，本文编号：2185748
本文链接：https://www.wllwen.com/yixuelunwen/yufangyixuelunwen/2185748.html
