本文選題：朊蛋白 + 抑郁癥��； 參考：《華東師范大學(xué)》2012年碩士論文

【摘要】：目的：研究糖皮質(zhì)激素引起神經(jīng)損傷的作用機(jī)制,主要研究細(xì)胞內(nèi)朊蛋白(Cellular prion protein, PrPc)是否參與到糖皮質(zhì)激素(Glucocorticoid, GC所造成的神經(jīng)損傷過(guò)程之中,初步探索PrPc可能的作用途徑。從而進(jìn)一步揭示抑郁癥的發(fā)病機(jī)制與治療方法。 方法：取新生24h內(nèi)的SD大鼠海馬組織,進(jìn)行海馬神經(jīng)細(xì)胞的原代培養(yǎng),運(yùn)用聚合酶鏈反應(yīng)(Polymerase chain reaction, PCR)、蛋白質(zhì)印跡(Western Blot)免疫熒光(Immuno fluorescence)、單核細(xì)胞直接細(xì)胞毒性測(cè)定(mono-nuclear cell direct cytotoxicity assay, MTT)等方法檢測(cè)GC對(duì)原代海馬細(xì)胞的損傷及相應(yīng)蛋白的變化；慢性應(yīng)激方法建立SD大鼠抑郁癥動(dòng)物模型后,運(yùn)用Open-field,強(qiáng)迫游泳等方法測(cè)試建模是否成功,運(yùn)用Western Blot、MTT等方法檢測(cè)慢性應(yīng)激抑郁癥大鼠與正常對(duì)照組大鼠腦中不同區(qū)域組織(海馬,皮層,中腦,紋狀體)中PrPc表達(dá)量的差別；運(yùn)用MTT方法檢測(cè)GC對(duì)人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y)的活性影響,運(yùn)用Western Blot方法檢測(cè)不同時(shí)間GC作用后,SH-SY5Y內(nèi)PrPc表達(dá)的變化,以及長(zhǎng)期GC作用后的SH-SY5Y是否更易受到NMDA的損傷；運(yùn)用病毒感染RNA干擾技術(shù)使PrPc表達(dá)下調(diào)(Gene knock-down)、通過(guò)Western Blot方法檢測(cè)PrPc表達(dá)下調(diào)的效率；用MTT方法檢測(cè)PrPc下調(diào)的SH-SY5Y細(xì)胞系與野生型SH-SY5Y細(xì)胞系受NMDA興奮性損傷的差異。 結(jié)果：SD大鼠原代海馬神經(jīng)細(xì)胞經(jīng)GC處理后NR2A、NR2B表達(dá)量提高,GR表達(dá)量明顯下調(diào),MR表達(dá)量下調(diào)不明顯,PrPc表達(dá)量下調(diào)；運(yùn)用慢性應(yīng)激建立的SD大鼠抑郁癥模型的海馬、皮層及紋狀體組織PrPc表達(dá)量下調(diào)；GC作用不同時(shí)間,SH-SY5Y細(xì)胞系內(nèi)的PrPc表達(dá)量先升高后下降,長(zhǎng)期GC作用后,細(xì)胞系更易受NMDA損傷。SiPrP與SiLuc田胞系相比,PrP knock-down的細(xì)胞有更易受到NMDA損傷的趨勢(shì),并且在某些濃度的NMDA作用時(shí)出現(xiàn)顯著差異。 結(jié)論：長(zhǎng)期GC作用可引起神經(jīng)細(xì)胞內(nèi)PrPc表達(dá)量的下調(diào),可能因此而導(dǎo)致NMDAR的激活,從而造成神經(jīng)細(xì)胞的興奮性損傷。
[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.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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