糖皮质激素引起神经损伤的机制研究

发布时间：2018-05-15 17:01

本文选题：朊蛋白 + 抑郁症　；参考：《华东师范大学》2012年硕士论文

【摘要】：目的：研究糖皮质激素引起神经损伤的作用机制,主要研究细胞内朊蛋白(Cellular prion protein, PrPc)是否参与到糖皮质激素(Glucocorticoid, GC所造成的神经损伤过程之中,初步探索PrPc可能的作用途径。从而进一步揭示抑郁症的发病机制与治疗方法。方法：取新生24h内的SD大鼠海马组织,进行海马神经细胞的原代培养,运用聚合酶链反应(Polymerase chain reaction, PCR)、蛋白质印迹(Western Blot)免疫荧光(Immuno fluorescence)、单核细胞直接细胞毒性测定(mono-nuclear cell direct cytotoxicity assay, MTT)等方法检测GC对原代海马细胞的损伤及相应蛋白的变化；慢性应激方法建立SD大鼠抑郁症动物模型后,运用Open-field,强迫游泳等方法测试建模是否成功,运用Western Blot、MTT等方法检测慢性应激抑郁症大鼠与正常对照组大鼠脑中不同区域组织(海马,皮层,中脑,纹状体)中PrPc表达量的差别；运用MTT方法检测GC对人神经母细胞瘤细胞(SH-SY5Y)的活性影响,运用Western Blot方法检测不同时间GC作用后,SH-SY5Y内PrPc表达的变化,以及长期GC作用后的SH-SY5Y是否更易受到NMDA的损伤；运用病毒感染RNA干扰技术使PrPc表达下调(Gene knock-down)、通过Western Blot方法检测PrPc表达下调的效率；用MTT方法检测PrPc下调的SH-SY5Y细胞系与野生型SH-SY5Y细胞系受NMDA兴奋性损伤的差异。结果：SD大鼠原代海马神经细胞经GC处理后NR2A、NR2B表达量提高,GR表达量明显下调,MR表达量下调不明显,PrPc表达量下调；运用慢性应激建立的SD大鼠抑郁症模型的海马、皮层及纹状体组织PrPc表达量下调；GC作用不同时间,SH-SY5Y细胞系内的PrPc表达量先升高后下降,长期GC作用后,细胞系更易受NMDA损伤。SiPrP与SiLuc田胞系相比,PrP knock-down的细胞有更易受到NMDA损伤的趋势,并且在某些浓度的NMDA作用时出现显著差异。结论：长期GC作用可引起神经细胞内PrPc表达量的下调,可能因此而导致NMDAR的激活,从而造成神经细胞的兴奋性损伤。
[Abstract]:Objective: To study the mechanism of glucocorticoid induced nerve injury and to investigate whether the Cellular prion protein (PrPc) is involved in the process of nerve injury caused by Glucocorticoid (Glucocorticoid, GC), and to explore the possible pathway of the possible action of PrPc, thus further revealing the pathogenesis and treatment of depression. Therapy.
Methods: the hippocampal tissues of SD rats in new 24h were taken to carry out primary culture of hippocampal neurons, using polymerase chain reaction (Polymerase chain reaction, PCR), immunofluorescence (Immuno fluorescence) of protein imprinting (Immuno fluorescence) and direct cytotoxicity of mononuclear cells (mono-nuclear cell). Methods the damage of primary hippocampal cells and the changes of corresponding proteins were detected by GC. After the chronic stress method established the SD rat model of depression, the methods of Open-field, forced swimming and other methods were used to test the success of the modeling. Western Blot, MTT and other methods were used to detect the different regions of the brain of the chronic stress depressive rats and the normal control rats. The difference in the expression of PrPc in the tissue (hippocampus, cortex, midbrain, striatum); the effect of GC on the activity of GC on human neuroblastoma cells (SH-SY5Y), and the changes in PrPc expression in SH-SY5Y after GC action at different time, and whether SH-SY5Y after the action of long-term GC were more vulnerable to NMDA damage by the Western Blot method. PrPc expression was downregulated (Gene knock-down) by virus infection RNA interference technique, and the efficiency of PrPc downregulation was detected by Western Blot method, and MTT method was used to detect the difference between SH-SY5Y cell line and wild type SH-SY5Y cell line of PrPc down-regulation by NMDA excitatory damage.
Results: after GC treatment, the expression of NR2A and NR2B in the primary hippocampal neurons of SD rats increased, the expression of GR was obviously down, the expression of MR was down regulated and the expression of PrPc was down regulated. The PrPc table of the hippocampus, cortex and striatum in the depressive model of SD rats was down regulated by chronic stress; GC action was in different time and SH-SY5Y cell line. The expression of PrPc was increased first and then decreased. After long-term GC, the cell line was more susceptible to NMDA damage and compared with the SiLuc field, PrP knock-down cells were more vulnerable to NMDA damage, and there were significant differences in some concentrations of NMDA.
Conclusion: long term GC can cause the downregulation of PrPc expression in neurons, which may lead to NMDAR activation, resulting in excitability injury of nerve cells.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】