糖皮质激素和细胞因子与海马突触损害的关系及抗炎细胞因子IL-10的保护作用

发布时间：2018-05-10 18:31

本文选题：糖皮质激素 + 海马　；参考：《华中科技大学》2006年博士论文

【摘要】： 第一部分慢性给予应激水平的糖皮质激素对海马神经元和突触的影响为探讨慢性给予应激水平糖皮质激素(glucocorticoid)对大鼠海马的影响，，在实验组大鼠饮用水中加入皮质醇，使动物皮质醇吸收量达10mg／kg／day，从而引起与应激动物相同的皮质醇水平，共21天，然后用免疫组织化学和Western blot的方法观察海马Caspase-3、Synaptophysin(Syn)和Neurogranin(Ng)表达的变化。结果显示：①与正常组相比，实验组大鼠海马结构中可见较多的细胞萎缩，海马各CA区和DG区未见或偶见极个别Caspase-3免疫反应阳性细胞，但在下托和压部后皮质中，可见有较多的Caspase-3免疫反应阳性细胞；②与正常组Syn和Ng表达水平(1.2197±0.3443和1.9330±0.3450)相比，实验组大鼠海马结构中Syn和Ng的表达明显减少(P＜0.05)，其表达水平分别为0.5512±0.0540和1.3375±0.3317。上述结果表明，慢性给予应激水平的糖皮质激素可损伤海马的功能，引起不依赖Caspase-3的海马神经细胞退变、使海马突触数量减少以及改变海马突触效能可能是其作用的部分机制。第二部分胸腺缺陷与海马损害的关系研究目的：探讨胸腺缺陷与海马损害的关系及其可能作用机制。方法：随机选取10～12周龄、22～24周龄的雄性裸小鼠(Balb／c-nu／nu)各6只作实验组，以同周龄、同数量、同性别的正常Balb／c小鼠作对照组，用ELISA方法检测小鼠血清和海马促炎细胞因子IL-2、IFN-γ、TNF-α的含量和Western blot方法分析海马突触蛋白Synaptophysin(Syn)、Neurogranin(Ng)的表达水平。结果：①10～12周龄实验组小鼠血清促炎细胞因子IFN-γ和TNF-α含量分别为184.98±81.69pg／ml和113.16±6.60pg／ml，显著高于同周龄对照组的71.81±9.76pg／ml(P＜0.05)和91.38±6.14(P＜0.01)；②22～24周龄实验组小鼠海马促炎细胞因子IFN-γ含量(6.97±0.64pg／mg海马组织)显著高于同周龄对照组(5.72±0.31pg／mg海马组织)(P＜0.01)：③22～24周龄实验组小鼠海马Syn表达水平显著低于同周龄对照组(对照组为0.7044±0.0882，实验组为0.5513±0.1096，P＜0.05)，并伴有Ng表达水平的升高(对照组为1.3030±0.2607，实验组为1.7484±0.2191，P＜0.01)。结论：胸腺缺陷会导致海马功能的损害，其机制可能与其所致的促炎细胞因子水平增高有关。第三部分抗炎细胞因子IL-10对糖皮质激素所致海马突触损害的保护作用目的探讨GC是否可通过引起促炎细胞因子作用增强来损害海马，以及抗炎细胞因子IL-10对GC所致海马损害的保护作用。方法①在原代培养的海马细胞中给予皮质醇8×10~(-6)mol／L，12h后用RT-PCR的方法观察其对海马细胞促炎细胞因子IFN-γ表达的影响；②构建IL-10真核表达质粒，并将其通过侧脑室插管注射到正通过饮水给予皮质醇(40mg/L，共21d)的Wistar大鼠脑中(80ng/20μl，每间隔4d注射1次，即在第1，5，9，13和17天注射)，然后用Western blot的方法观察其对海马突触蛋白Synapsin I和Neurogranin(Ng)表达的影响。结果①皮质醇可引起原代培养的海马细胞促炎细胞因子IFN-γmRNA表达增加：皮质醇组IFN-γmRNA表达水平为0.5940+0.0448(n=4)，对照组为0.4237+0.0915(n=4)，p＜0.05；②侧脑室给予IL-10的实验组大鼠Synapsin I和Ng的表达水平分别为2.0171±0.2527、1.9653+0.0843(n=7)，显著高于对照组的1.1440±0.2152、1.5344±0.1763(n=7)，p＜0.01。结论促炎细胞因子可能参与了糖皮质激素对海马的损害，IL-10对糖皮质激素所致的海马损害有保护作用。
[Abstract]:Part 1 the effects of chronic stress levels of glucocorticoids on hippocampal neurons and synapses
To investigate the effect of Glucocorticoid (glucocorticoid) on the hippocampus of rats with chronic stress level, cortisol was added to the drinking water of rats in the experimental group to reach 10mg / kg / day, resulting in the same cortisol level as the stress animal, for 21 days, and then observed by immunohistochemistry and Western blot. The changes in the expression of Caspase-3, Synaptophysin (Syn) and Neurogranin (Ng) in the hippocampus showed that: 1. Compared with the normal group, there were more cell atrophy in the hippocampus of the experimental group, and there was no or even a few Caspase-3 immunoreactive fine cells in the CA and DG regions of the hippocampus, but there were more Casp in the lower and the posterior cortex. Compared with the expression level of Syn and Ng (1.2197 + 0.3443 and 1.9330 + 0.3450) in the normal group (1.2197 + 0.3443 and 1.9330 + 0.3450), the expression of Syn and Ng in the hippocampus of the experimental group was significantly reduced (P < 0.05). The expression levels were 0.5512 + 0.0540 and 1.3375 + 0.3317., respectively, indicating that the chronic stress level of glucocorticoid could be found. Damage to the hippocampus, which causes degeneration of hippocampal neurons that do not depend on Caspase-3, may be a part of the mechanism of the effects of the reduction of the hippocampal synapse and the change of the hippocampal synaptic efficacy.
The second part is the relationship between thymus defect and hippocampus damage.
Objective: To investigate the relationship and possible mechanism of thymus defects and hippocampus damage. Methods: 6 male nude mice (Balb / c-nu / nu) were randomly selected as experimental groups at the age of 10~12 weeks and 22~24 weeks old, with the same week age, the same number and same sex normal Balb / c mice, and ELISA method was used to detect the serum and hippocampus of mice. The content of cytokine IL-2, IFN- gamma, TNF- alpha and Western blot method were used to analyze the expression level of Synaptophysin (Syn) and Neurogranin (Ng) in hippocampal synaptic protein. Results: (1) the levels of serum proinflammatory cytokines IFN- gamma and TNF- alpha in the serum of 10~12 weeks old mice were 184.98 + 81.69pg / TNF- and 113.16 +, respectively, which were significantly higher than those of the same age group. 71.81 + 9.76pg / ml (P < 0.05) and 91.38 + 6.14 (P < 0.01). The content of IFN- gamma (6.97 + 0.64pg / Mg in hippocampus) of hippocampus in 22~24 weeks old mice was significantly higher than that of the same week age group (5.72 + 0.31pg / Mg hippocampal tissue) (P < 0.01). 3. The Syn expression level in the hippocampus of 22~24 weeks old mice was significantly lower than that of the same group. The control group was 0.7044 + 0.0882, the experimental group was 0.5513 + 0.1096, P < 0.05), and the level of Ng expression increased (the control group was 1.3030 + 0.2607, the experimental group was 1.7484 + 0.2191, P < 0.01). Conclusion: the thymic defect may lead to the damage of the hippocampus function, and its mechanism may be related to the increase of the level of proinflammatory cytokines.
The third part is the protective effect of anti-inflammatory cytokine IL-10 on hippocampal synaptic damage induced by glucocorticoid.
Objective to investigate the protective effect of GC on hippocampal damage induced by the enhancement of the role of pro-inflammatory cytokines and the anti-inflammatory cytokine IL-10 on the hippocampal damage caused by GC. Methods 1. In the primary cultured hippocampal cells, 8 x 10~ (-6) mol / L was given in the hippocampus cells, and 12h was used to observe the IFN- gamma table of the hippocampal proinflammatory cytokines by RT-PCR method after 12h. The IL-10 eukaryotic expression plasmid was constructed and injected through the lateral ventricle intubation into the Wistar rat brain which was given 40mg/L (co 21d) by drinking water (80ng/20 Mu L, injected 1 times per interval 4D, that is, injected at 1,5,9,13 and 17 days), and then observed the Synapsin I and the hippocampal synapse protein by Western blot. The effect of ranin (Ng) expression. Results (1) cortisol could increase the expression of IFN- gamma mRNA in the primary cultured hippocampal cells: the level of IFN- gamma mRNA expression in cortisol group was 0.5940+0.0448 (n=4), the control group was 0.4237+0.0915 (n=4), P < 0.05. 2.0171 + 0.2527,1.9653+0.0843 (n=7), significantly higher than that of the control group, 1.1440 + 0.2152,1.5344 + 0.1763 (n=7), P < 0.01. conclusion proinflammatory cytokines may be involved in the damage of glucocorticoid to hippocampus, IL-10 has protective effect on hippocampal damage caused by glucocorticoid.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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