本文選題：砷暴露 + 神經(jīng)元再生��； 參考：《大連醫(yī)科大學(xué)》2013年博士論文

【摘要】：目的 砷暴露可以導(dǎo)致神經(jīng)系統(tǒng)的損傷。本研究的目的是觀察成年小鼠慢性砷暴露以后，大腦海馬的細胞增殖和神經(jīng)元再生能力是否受到影響；以及停止砷暴露后，海馬的細胞增殖和神經(jīng)元再生能力是否恢復(fù)正常。此外，為了探索砷誘導(dǎo)的中樞神經(jīng)系統(tǒng)毒性損傷機制，從核酸水平檢測其是否受到損傷，提供毒理學(xué)證據(jù)。 方法 在對海馬神經(jīng)元再生的研究中，將90只小鼠隨機分成3組。第1組小鼠飲用蒸餾水4個月（對照組）；第2組小鼠飲用含4.0mg/L As2O3的水4個月（染砷組）；第3組小鼠飲用含4.0mg/L As2O3的水2個月，然后改為蒸餾水，再飲用2個月（恢復(fù)組）。用電感耦合等離子體質(zhì)譜法對小鼠大腦和血清中的砷含量進行檢測；用HE染色觀察海馬組織病理學(xué)改變；用激光共聚焦顯微鏡鑒定海馬顆粒細胞層（granule cell layer，GCL）和顆粒細胞亞層（subgranular zone, SGZ）5-溴-2’-脫氧尿嘧啶核苷（5-Bromo-2’-deoxyUridine, BrdU）陽性的細胞作為增殖的標(biāo)志，BrdU+和NeuN+雙標(biāo)記細胞作為新生神經(jīng)元的標(biāo)志；用末端脫氧核苷酸轉(zhuǎn)移酶dUTP缺口末端標(biāo)記測定法(Terminal deoxynucleotidyl transferase dUTP nick end-labeling assay,TUNEL)檢測該區(qū)細胞凋亡；用Fluoro-Jade C檢測該區(qū)細胞的死亡；用Real-timePCR檢測各組海馬超氧化物歧化酶（superoxide dismutase, SOD）1、SOD2及Wnt3基因的表達差異；用Morris水迷宮實驗評價各組小鼠的空間學(xué)習(xí)記憶能力。 在對腦組織損傷的研究中，32只小鼠被隨機分成4組，自由飲用0，1,2和4mg/L As2O3水，觀察了腦組織的病理變化，并用免疫組化的方法檢測了小鼠腦8-NO2-G的表達。 結(jié)果 1、小鼠血清和大腦砷含量 經(jīng)過2個月的砷暴露，對照組小鼠血清和大腦中的砷濃度分別為28±9.4和31±5.0ng/g；染砷組小鼠血清和大腦中的砷濃度分別為42±6.2和60±8.5ng/g，染砷組小鼠大腦中的砷含量顯著高于對照組(p 0.05)。再經(jīng)過2個月的砷暴露，樣本中的砷含量繼續(xù)升高，染砷組小鼠血清和大腦中的砷濃度分別為61±3.2和74±6.3ng/g，顯著高于對照組小鼠血清和大腦中的砷濃度（31±3.5和22±2.4ng/g，p 0.01)，而恢復(fù)組血清和大腦中的砷濃度分別為25±4.4和23±2.8ng/g，與染砷組相比顯著降低(p0.01)，與對照組相比無顯著差異。以上結(jié)果提示，砷暴露以后，砷可以在腦中沉積，當(dāng)砷從飲水中去除，沉積的砷可以排泄到體外。 2、海馬的組織病理學(xué)改變 組織病理學(xué)觀察發(fā)現(xiàn)，砷暴露后小鼠海馬神經(jīng)元出現(xiàn)病理損傷。對照組（2個月、4個月）均無異常病理改變。經(jīng)過4個月的砷暴露，染砷組出現(xiàn)明顯的核固縮，而恢復(fù)組海馬有顯著的改善。2個月染砷組海馬神經(jīng)元的病理損傷相對較輕。 3、砷對細胞增殖的抑制及其恢復(fù) 經(jīng)過2個月的砷暴露，SGZ/GCL范圍內(nèi)，BrdU+細胞的數(shù)量是104.2±5.4個細胞/切片，比對照組(120.7±12.6個細胞/切片)降低，但無顯著差異。經(jīng)過4個月的砷暴露，對照組、染砷組和恢復(fù)組BrdU+細胞的數(shù)量分別為111.8±5.6、84.0±2.3和128.5±4.8個細胞/切片。與對照組相比，染砷組BrdU+細胞的數(shù)量顯著降低(p0.01)，表明砷暴露抑制了細胞的增殖。與染砷組相比，恢復(fù)組BrdU+細胞的數(shù)量顯著升高(p 0.01)，恢復(fù)組與對照組無差別，提示飲水中的砷改為蒸餾水，恢復(fù)了SGZ/GCL中細胞的增殖。 4、砷對神經(jīng)元再生的抑制及其恢復(fù) 新的成熟的海馬神經(jīng)元（神經(jīng)元再生）表現(xiàn)為SGZ/GCL中細胞BrdU和NeuN雙標(biāo)記陽性。砷暴露2個月后，染砷組與對照組相比，BrdU+和NeuN+雙標(biāo)記細胞占BrdU+細胞百分比顯著降低(54±2.7vs.67±2.3%, p0.01)。4個月時，恢復(fù)組BrdU+和NeuN+雙標(biāo)記細胞占BrdU+細胞百分比顯著高于染砷組(71±2.2vs.60±2.8%,p0.05),并與對照組相比沒有差別(71±2.2vs.73±1.7%, p0.05)。 5、各組間凋亡及死亡無顯著差別 每只小鼠SGZ/GCL中計數(shù)至少1000個Hoechst染色陽性細胞。經(jīng)過2個月的砷暴露，TUNEL陽性細胞百分比：對照組為0.58±0.075%，染砷組為0.65±0.077%，組間沒有顯著差別（p0.05）。將砷改成蒸餾水2個月后，TUNEL陽性細胞百分比：對照組為0.67±0.065%，染砷組為0.77±0.063%，恢復(fù)組為0.75±0.087%，各組間沒有顯著差別（p0.05）。每只小鼠SGZ/GCL中計數(shù)至少1000個細胞，未見Fluoro-Jade C陽性細胞。 6、海馬SOD1、SOD2及Wnt3的mRNA表達 經(jīng)過2個月的砷暴露，染砷組SOD1/β-actin mRNA比值是0.42±0.0058，較對照組（0.65±0.013）顯著降低(p0.01)。經(jīng)過4個月的砷暴露，，對照組、染砷組和恢復(fù)組SOD1/β-actin mRNA比值分別為0.61±0.017,0.40±0.0088和0.56±0.0088。與對照組相比，染砷組SOD1表達顯著降低(p0.01)，在恢復(fù)組中，SOD1的水平與染砷組相比顯著提高(p0.01)，但并沒有恢復(fù)至對照組水平(p0.05)。 經(jīng)過2個月的砷暴露，染砷組SOD2/β-actin mRNA比值是0.29±0.015×10-1，較對照組0.45±0.0033×10-1顯著降低(p0.01)。經(jīng)過4個月的砷暴露，對照組、染砷組和恢復(fù)組SOD2/β-actin mRNA比值分別為0.60±0.012×10-1,0.42±0.012×10-1和0.54±0.0088×10-1。與對照組相比，染砷組SOD2表達顯著降低(p0.01)，在恢復(fù)組中，SOD2的水平與染砷組相比顯著提高(p0.01)，但并沒有恢復(fù)至對照組水平(p0.01)。 經(jīng)過4個月的砷暴露，染砷組Wnt3/β-actin mRNA比值較對照組降低(p0.01)，但在恢復(fù)組中，Wnt3的水平并沒有恢復(fù)。 7、Morris水迷宮實驗 在隱藏平臺獲得實驗及空間搜索實驗中，經(jīng)過2個月的砷暴露，各組間無顯著差異。經(jīng)過4個月的砷暴露，各組間依然無顯著差異。 8、8-硝基鳥嘌呤的檢測 經(jīng)過2個月的砷暴露，用免疫組化的方法，檢測出小鼠腦組織中8-硝基鳥嘌呤的高表達，同時伴有組織病理學(xué)的改變，提示細胞中核酸的氧化及硝化損傷。 結(jié)論 1、慢性砷暴露可以抑制小鼠大腦海馬細胞的增殖和神經(jīng)元再生。 2、該抑制在停止砷暴露后可以恢復(fù)。 3、亞慢性砷暴露小鼠腦組織中有8-硝基鳥嘌呤的高表達。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R114;Q42

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