内源性ROS对小鼠脑发育的影响

发布时间：2018-03-20 02:40

本文选题：活性氧　切入点：N-乙酰半胱氨酸　出处：《南京医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景和目的：活性氧(reactive oxygen species, ROS)是线粒体氧化代谢过程中产生的具有高度活性的一类化学物质,参与调控生物体的诸多生理功能。本学位论文旨在通过对孕期和哺乳期母鼠给予抗氧化剂N-乙酰半胱氨酸(N-acetyl-cysteine, NAC)处理,观察其对子代出生后脑生长和发育的影响。材料和方法：二月龄C57BL/6背景小鼠按照1雄鼠：2雌鼠的比例进行合笼后,再将小鼠随机分为2组：NAC给药组(饮水中添加NAC6.523g/L)和正常对照组。取子代小鼠于P1、P3、P7、P14、P21进行下列比较研究：(1)测量其体重、脑重,观察整体与脑的大体发育状况；(2)尼氏染色观察脑组织学发育变化；(3)免疫组织化学染色检测增殖细胞核抗原(PCNA)的表达,以观察脑内神经细胞增殖情况(4)免疫组织化学染色检测神经元核蛋白(NeuN)、胶质纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)的表达,以明确脑内神经细胞的分化情况；(5)取P3小鼠,腹腔内注射二氢乙啶4小时后,采用冰冻切片,荧光显微镜下检测小鼠脑内ROS水平。结果：(1)P1、P3,两组小鼠的体重和脑重及外形并无显著差异；而在P7、P14,给药组小鼠体重和脑重轻于正常组；至P21,两者之间又无明显差异。(2)尼氏染色结果显示,在P1、P3,两组小鼠的脑嘴尾径并无明显差异；但在P7和P14天,NAC组小鼠脑小于正常对照组；至P21,两者之间无显著性差异。此外,正常小鼠CA锥状细胞层和DG的颗粒细胞层面积从P1-P7逐渐增大,至P7,海马的大小和外形与P14和P21趋于一致；而NAC给药组小鼠在P3和P7明显小于正常小鼠,其他各时间点与对照组趋于一致。NAC给药组小鼠小脑在P1、P3并无明显差异,在P7-P21小脑截面积稍小于正常对照组,但无统计学意义。但至P21,NAC小鼠小脑各叶的叶片发育仍稍逊于正常对照组。(3)两组小鼠在P1大脑皮层、海马和小脑内均有PCNA免疫阳性细胞表达,至P3达到高峰后逐渐下调。在P3,NAC组上述脑区PCNA阳性细胞数量和染色强度要低于对照组。(4)Neun染色结果显示,在出生后脑内逐渐表达增强。在p 3, NAC组小鼠的大脑皮层、海马内Neun阳性细胞染色强度与数量均低于对照组,但在小脑两者之间的差异不明显；其余各时间点两者之间无明显差异。NAC组小鼠海马和小脑皮质内GFAP表达水平在P3和P7低于对照组,其他各时间点差异不明显。正常小鼠小脑在P3开始出现少量MBP阳性纤维,但NAC组小鼠未见表达。而前脑在P14才出现MBP免疫阳性信号,且NAC组的表达强度低于对照组小鼠。(5)二氢乙啶荧光染色结果显示NAC组小鼠海马及小脑ROS水平明显低于对照组。结论：孕期和哺乳期给予抗氧化剂NAC,导致子代小鼠出生后脑大小和重量增长、神经前体细胞增殖以及向神经元、星形胶质细胞和少突胶质细胞的分化时程一过性延迟。这些实验结果提示,内源性ROS对脑发育是不可或缺的,孕期和哺乳期不恰当的添加抗氧化剂有可能影响脑发育的正常进程。
[Abstract]:Background and objective: reactive oxygen species (Ros) are highly active chemicals produced during mitochondrial oxidative metabolism. This dissertation aims to regulate the physiological functions of organisms through the administration of the antioxidant N-acetyl-cysteine (NACC-) on pregnant and lactating mothers. Materials and methods: two month old C57BL / 6 background mice were caged in proportion of 1 male to 2 female mice. Then the mice were randomly divided into two groups: the control group (supplemented with NAC 6.523g / L in drinking water) and the control group. To observe the gross development of the whole and the brain, the expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical staining. The expression of neuronal nuclear protein (Neu Nu), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) were detected by immunohistochemical staining to determine the differentiation of neurons in brain. After intraperitoneal injection of dihydroethidime for 4 hours, frozen sections were used to detect the ROS level in the brain of mice. Results there was no significant difference in body weight, brain weight and appearance between the two groups. However, in P7 P14, the body weight and brain weight of the mice in the administration group were lighter than those in the normal group, but there was no significant difference between the two groups in the body weight and brain weight at P21.The results showed that there was no significant difference in the brain mouth and tail diameters between the two groups. However, the brain of NAC group was smaller than that of normal control group on P7 and P14 days, but there was no significant difference between them at P21. In addition, the area of CA pyramidal cell layer and granular cell layer of DG in normal mice increased from P1-P7 to P1-P7. At P7, the hippocampal size and shape were similar to those of P14 and P21, while those in NAC group were significantly lower than those in normal mice, and there was no significant difference between the other time points and the control group in the cerebellum of the mice treated with P14 and P21. The cerebellar cross-sectional area of P7-P21 was slightly smaller than that of the normal control group, but there was no statistical significance. However, the leaf development of the cerebellar lobes of P21 NAC mice was still slightly inferior to that of the normal control group.) in the P1 cerebral cortex, hippocampus and cerebellum, the expression of PCNA immunoreactive cells was found in the cortex, hippocampus and cerebellum of the two groups. The number and intensity of PCNA positive cells in the above mentioned brain regions in the P3 + NAC group were lower than those in the control group. The results showed that the expression increased gradually in the postnatal brain, and increased gradually in the cerebral cortex of the mice in the p3 + NAC group. The staining intensity and quantity of Neun positive cells in hippocampus were lower than those in control group, but there was no significant difference between them. The expression of GFAP in hippocampus and cerebellar cortex in NAC group was lower than that in control group, but there was no significant difference at other time points. A small amount of MBP positive fibers began to appear in cerebellum of normal mice at P3. However, no expression was found in NAC mice, and MBP immunopositive signals appeared in the forebrain at P14. The expression of ROS in hippocampus and cerebellum in NAC group was lower than that in control group. Conclusion: during pregnancy and lactation, the level of ROS in hippocampus and cerebellum of mice in NAC group was significantly lower than that in control group. The brain grows in size and weight after birth, The proliferation of neural precursor cells and the transient delay in differentiation into neurons, astrocytes and oligodendrocytes. These results suggest that endogenous ROS is essential for brain development. Improper supplementation of antioxidants during pregnancy and lactation may affect the normal course of brain development.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R338

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