砷对小鼠海马神经元再生及脑组织损伤的研究

发布时间：2018-05-23 07:38

本文选题：砷暴露 + 神经元再生　；参考：《大连医科大学》2013年博士论文

【摘要】：目的砷暴露可以导致神经系统的损伤。本研究的目的是观察成年小鼠慢性砷暴露以后，大脑海马的细胞增殖和神经元再生能力是否受到影响；以及停止砷暴露后，海马的细胞增殖和神经元再生能力是否恢复正常。此外，为了探索砷诱导的中枢神经系统毒性损伤机制，从核酸水平检测其是否受到损伤，提供毒理学证据。方法在对海马神经元再生的研究中，将90只小鼠随机分成3组。第1组小鼠饮用蒸馏水4个月（对照组）；第2组小鼠饮用含4.0mg/L As2O3的水4个月（染砷组）；第3组小鼠饮用含4.0mg/L As2O3的水2个月，然后改为蒸馏水，再饮用2个月（恢复组）。用电感耦合等离子体质谱法对小鼠大脑和血清中的砷含量进行检测；用HE染色观察海马组织病理学改变；用激光共聚焦显微镜鉴定海马颗粒细胞层（granule cell layer，GCL）和颗粒细胞亚层（subgranular zone, SGZ）5-溴-2’-脱氧尿嘧啶核苷（5-Bromo-2’-deoxyUridine, BrdU）阳性的细胞作为增殖的标志，BrdU+和NeuN+双标记细胞作为新生神经元的标志；用末端脱氧核苷酸转移酶dUTP缺口末端标记测定法(Terminal deoxynucleotidyl transferase dUTP nick end-labeling assay,TUNEL)检测该区细胞凋亡；用Fluoro-Jade C检测该区细胞的死亡；用Real-timePCR检测各组海马超氧化物歧化酶（superoxide dismutase, SOD）1、SOD2及Wnt3基因的表达差异；用Morris水迷宫实验评价各组小鼠的空间学习记忆能力。在对脑组织损伤的研究中，32只小鼠被随机分成4组，自由饮用0，1,2和4mg/L As2O3水，观察了脑组织的病理变化，并用免疫组化的方法检测了小鼠脑8-NO2-G的表达。结果 1、小鼠血清和大脑砷含量经过2个月的砷暴露，对照组小鼠血清和大脑中的砷浓度分别为28±9.4和31±5.0ng/g；染砷组小鼠血清和大脑中的砷浓度分别为42±6.2和60±8.5ng/g，染砷组小鼠大脑中的砷含量显著高于对照组(p 0.05)。再经过2个月的砷暴露，样本中的砷含量继续升高，染砷组小鼠血清和大脑中的砷浓度分别为61±3.2和74±6.3ng/g，显著高于对照组小鼠血清和大脑中的砷浓度（31±3.5和22±2.4ng/g，p 0.01)，而恢复组血清和大脑中的砷浓度分别为25±4.4和23±2.8ng/g，与染砷组相比显著降低(p0.01)，与对照组相比无显著差异。以上结果提示，砷暴露以后，砷可以在脑中沉积，当砷从饮水中去除，沉积的砷可以排泄到体外。 2、海马的组织病理学改变组织病理学观察发现，砷暴露后小鼠海马神经元出现病理损伤。对照组（2个月、4个月）均无异常病理改变。经过4个月的砷暴露，染砷组出现明显的核固缩，而恢复组海马有显著的改善。2个月染砷组海马神经元的病理损伤相对较轻。 3、砷对细胞增殖的抑制及其恢复经过2个月的砷暴露，SGZ/GCL范围内，BrdU+细胞的数量是104.2±5.4个细胞/切片，比对照组(120.7±12.6个细胞/切片)降低，但无显著差异。经过4个月的砷暴露，对照组、染砷组和恢复组BrdU+细胞的数量分别为111.8±5.6、84.0±2.3和128.5±4.8个细胞/切片。与对照组相比，染砷组BrdU+细胞的数量显著降低(p0.01)，表明砷暴露抑制了细胞的增殖。与染砷组相比，恢复组BrdU+细胞的数量显著升高(p 0.01)，恢复组与对照组无差别，提示饮水中的砷改为蒸馏水，恢复了SGZ/GCL中细胞的增殖。 4、砷对神经元再生的抑制及其恢复新的成熟的海马神经元（神经元再生）表现为SGZ/GCL中细胞BrdU和NeuN双标记阳性。砷暴露2个月后，染砷组与对照组相比，BrdU+和NeuN+双标记细胞占BrdU+细胞百分比显著降低(54±2.7vs.67±2.3%, p0.01)。4个月时，恢复组BrdU+和NeuN+双标记细胞占BrdU+细胞百分比显著高于染砷组(71±2.2vs.60±2.8%,p0.05),并与对照组相比没有差别(71±2.2vs.73±1.7%, p0.05)。 5、各组间凋亡及死亡无显著差别每只小鼠SGZ/GCL中计数至少1000个Hoechst染色阳性细胞。经过2个月的砷暴露，TUNEL阳性细胞百分比：对照组为0.58±0.075%，染砷组为0.65±0.077%，组间没有显著差别（p0.05）。将砷改成蒸馏水2个月后，TUNEL阳性细胞百分比：对照组为0.67±0.065%，染砷组为0.77±0.063%，恢复组为0.75±0.087%，各组间没有显著差别（p0.05）。每只小鼠SGZ/GCL中计数至少1000个细胞，未见Fluoro-Jade C阳性细胞。 6、海马SOD1、SOD2及Wnt3的mRNA表达经过2个月的砷暴露，染砷组SOD1/β-actin mRNA比值是0.42±0.0058，较对照组（0.65±0.013）显著降低(p0.01)。经过4个月的砷暴露，，对照组、染砷组和恢复组SOD1/β-actin mRNA比值分别为0.61±0.017,0.40±0.0088和0.56±0.0088。与对照组相比，染砷组SOD1表达显著降低(p0.01)，在恢复组中，SOD1的水平与染砷组相比显著提高(p0.01)，但并没有恢复至对照组水平(p0.05)。经过2个月的砷暴露，染砷组SOD2/β-actin mRNA比值是0.29±0.015×10-1，较对照组0.45±0.0033×10-1显著降低(p0.01)。经过4个月的砷暴露，对照组、染砷组和恢复组SOD2/β-actin mRNA比值分别为0.60±0.012×10-1,0.42±0.012×10-1和0.54±0.0088×10-1。与对照组相比，染砷组SOD2表达显著降低(p0.01)，在恢复组中，SOD2的水平与染砷组相比显著提高(p0.01)，但并没有恢复至对照组水平(p0.01)。经过4个月的砷暴露，染砷组Wnt3/β-actin mRNA比值较对照组降低(p0.01)，但在恢复组中，Wnt3的水平并没有恢复。 7、Morris水迷宫实验在隐藏平台获得实验及空间搜索实验中，经过2个月的砷暴露，各组间无显著差异。经过4个月的砷暴露，各组间依然无显著差异。 8、8-硝基鸟嘌呤的检测经过2个月的砷暴露，用免疫组化的方法，检测出小鼠脑组织中8-硝基鸟嘌呤的高表达，同时伴有组织病理学的改变，提示细胞中核酸的氧化及硝化损伤。结论 1、慢性砷暴露可以抑制小鼠大脑海马细胞的增殖和神经元再生。 2、该抑制在停止砷暴露后可以恢复。 3、亚慢性砷暴露小鼠脑组织中有8-硝基鸟嘌呤的高表达。
[Abstract]:objective
Arsenic exposure can cause nervous system damage. The purpose of this study was to observe whether cell proliferation and neuronal regeneration in the hippocampus were affected by chronic arsenic exposure in adult mice, and whether the cell proliferation and regeneration of the hippocampus were restored to normal after arsenic exposure was stopped. The mechanism of the central nervous system toxicity is to detect whether it is damaged by nucleic acid level and provide toxicological evidence.
Method
In the study of hippocampal regeneration, 90 mice were randomly divided into 3 groups. First groups of mice drank distilled water for 4 months (control group); second mice drank water containing 4.0mg/L As2O3 for 4 months (arsenic contamination group); the third mice drank water containing 4.0mg/L As2O3 for 2 months, then changed into distilled water, and then drank for 2 months (recovery group). Inductively coupled, and so on. The arsenic content in the brain and serum of mice was detected by plasma mass spectrometry; the pathological changes in the hippocampus were observed by HE staining; the granule cell layer (GCL) and the granular cell sublayer (subgranular zone, SGZ) were identified by laser confocal microscopy, and the 5- bromine -2 '- deoxy uridine (5-Bromo-2' -deoxyUridine) was identified. BrdU) positive cells as a marker of proliferation, BrdU+ and NeuN+ double labeled cells as a sign of new neurons; use the terminal deoxynucleotidyl transferase dUTP nick end labeling method (Terminal deoxynucleotidyl transferase dUTP nick end-labeling assay, TUNEL) to detect the cell apoptosis. The expression of superoxide dismutase (SOD) 1, SOD2 and Wnt3 were detected by Real-timePCR, and the spatial learning and memory ability of each group was evaluated by the Morris water maze test.
In the study of brain tissue injury, 32 mice were randomly divided into 4 groups, free drinking 0,1,2 and 4mg/L As2O3 water. The pathological changes of brain tissue were observed and the expression of 8-NO2-G in the brain of the mice was detected by immunohistochemical method.
Result
1, arsenic content in serum and brain of mice
After 2 months of arsenic exposure, the arsenic concentration in the serum and brain of the control group was 28 + 9.4 and 31 + 5.0ng/g, respectively. The arsenic concentration in the serum and brain of the mice was 42 + 6.2 and 60 + 8.5ng/g respectively. The arsenic content in the brain of the arsenic infected mice was significantly higher than that of the control group (P 0.05). The arsenic exposure in the samples after 2 months was followed by arsenic content in the samples. The arsenic concentration in the serum and brain of the mice was 61 + 3.2 and 74 + 6.3ng/g, respectively. The arsenic concentration in the serum and brain of the control mice was significantly higher than that in the serum and brain of the control mice (31 + 3.5 and 22 + 2.4ng/g, P 0.01), while the arsenic concentration in the serum and brain of the recovery group was 25 + 4.4 and 23 + 2.8ng/g, respectively, compared with the control group (P0.01), and the control group. The above results suggest that arsenic can be deposited in the brain after arsenic exposure, and arsenic can be excreted in the body when arsenic is removed from drinking water.
2, histopathological changes in the hippocampus
Histopathological observation found that the hippocampal neurons in the mice were exposed to pathological damage after arsenic exposure. There was no abnormal pathological changes in the control group (2 months, 4 months). After 4 months of arsenic exposure, the arsenic exposure group had obvious nuclear condensation, while the hippocampus of the recovery group had a significant improvement in the pathological damage of the hippocampal neurons in the.2 months of arsenic exposure group.
3, the inhibition and recovery of arsenic on cell proliferation
After 2 months of arsenic exposure, the number of BrdU+ cells in the SGZ/GCL range was 104.2 + 5.4 cells / sections, which were lower than the control group (120.7 + 12.6 cells / slices), but no significant difference was found. After 4 months of arsenic exposure, the number of BrdU+ cells in the arsenic group and the recovery group were 111.8 + 5.6,84.0 + 2.3 and 128.5 + 4.8 cells / slices. Compared with the control group, the number of BrdU+ cells in the arsenic staining group decreased significantly (P0.01), indicating that arsenic exposure inhibited the proliferation of cells. Compared with the arsenic exposure group, the number of BrdU+ cells in the recovery group increased significantly (P 0.01), and there was no difference between the recovery group and the control group, suggesting that the arsenic in the drinking water was converted to steam distillate and the proliferation of cells in SGZ/GCL was restored.
4, the inhibition and recovery of arsenic on the regeneration of neurons
The new mature hippocampal neurons (neuron regeneration) showed BrdU and NeuN double labeled positive cells in SGZ/GCL. After 2 months of arsenic exposure, the percentage of BrdU+ and NeuN+ double labeled cells accounted for a significant decrease in the percentage of BrdU+ cells (54 + 2.7vs.67 + 2.3%, P0.01).4 months after arsenic exposure, and BrdU+ and NeuN+ double labeled cells in the recovery group were BrdU+ thin. The percentage of cells was significantly higher than that of arsenic group (71 + 2.2vs.60 + 2.8%, P0.05), and there was no difference (71 + 2.2vs.73 + 1.7%, P0.05) compared with the control group.
5, there was no significant difference in apoptosis and death among all groups
At least 1000 Hoechst positive cells were counted in each mouse SGZ/GCL. After 2 months of arsenic exposure, the percentage of TUNEL positive cells in the control group was 0.58 + 0.075% and the arsenic staining group was 0.65 + 0.077%. There was no significant difference between the groups (P0.05). The percentage of TUNEL positive cells was 0.67 + 0.065% in the control group after 2 months of distilled water, and the arsenic staining group was in the control group. The group was 0.77 + 0.063%, and the recovery group was 0.75 + 0.087%. There was no significant difference between each group (P0.05). At least 1000 cells were counted in each mouse SGZ/GCL, and no Fluoro-Jade C positive cells were found.
6, mRNA expression in hippocampal SOD1, SOD2 and Wnt3
After 2 months of arsenic exposure, the ratio of SOD1/ beta -actin mRNA in the arsenic staining group was 0.42 + 0.0058, compared with the control group (0.65 + 0.013) significantly (P0.01). After 4 months of arsenic exposure, the ratio of SOD1/ beta -actin mRNA in the arsenic group and the recovery group was 0.61 + 0.017,0.40 + 0.0088 and 0.56 + 0.0088. compared with the control group, and the expression of SOD1 in the arsenic staining group decreased significantly. In low recovery (P0.01), the level of SOD1 in the recovery group was significantly higher than that in the arsenic group (P0.01), but it did not return to the control group (P0.05).
After 2 months of arsenic exposure, the ratio of SOD2/ beta -actin mRNA in the arsenic staining group was 0.29 + 0.015 x 10-1, compared with 0.45 + 0.0033 x 10-1 in the control group (P0.01). After 4 months of arsenic exposure, the control group, the ratio of SOD2/ beta -actin mRNA in the arsenic and recovery groups was 0.60 + 0.012 * 10-1,0.42 + 0.012 x 10-1 and 0.54 + 0.0088 * 10-1., respectively, compared with the control group. The expression of SOD2 in arsenic group was significantly decreased (P0.01). In the recovery group, the level of SOD2 increased significantly compared with the arsenic group (P0.01), but it did not return to the control group (P0.01).
After 4 months of arsenic exposure, the ratio of Wnt3/ beta -actin mRNA in arsenic exposed group was lower than that in the control group (P0.01), but in the recovery group, the level of Wnt3 did not recover.
7, Morris water maze experiment
There was no significant difference between each group after 2 months of arsenic exposure in the experiment and space search experiment of hidden platform. After 4 months of arsenic exposure, there was still no significant difference between each group.
Detection of 8,8- nitro guanine
After 2 months of arsenic exposure, the high expression of 8- nitroguanine in the brain tissue of mice was detected by immunohistochemical method, accompanied by histopathological changes, suggesting the oxidative and nitrification damage of nucleic acids in the cells.
conclusion
1, chronic arsenic exposure can inhibit the proliferation and regeneration of hippocampal neurons in mice.
2, the inhibition can be recovered after arsenic exposure is stopped.
3, high levels of 8- nitroguanine were found in brain tissues of mice exposed to subchronic arsenic exposure.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114;Q42

【参考文献】