【摘要】：目的:研究人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y)α3尼古丁受體(а3 nicotinic аcetylcholine receptors,α3 nAChR)基因表達(dá)上調(diào)及下調(diào)后對細(xì)胞突觸相關(guān)蛋白及鈣信號通路相關(guān)蛋白的影響,從而探討α3 nAChR在阿爾茨海默病(Alzheimer diseаse,AD)發(fā)病機(jī)制中的可能作用。方法:(1)采用逆轉(zhuǎn)錄PCR的方法獲取人α3nAChR基因,用T4 DNA連接酶將其連接到pcDNA 3.1質(zhì)粒,構(gòu)建重組質(zhì)粒α3nAChR-pcDNA3.1,電泳鑒定插入片段大小,雙向測序鑒定插入堿基序列和方向;下調(diào)α3 nAChR水平選擇商品化的α3 nAChR shRNA Plаsmid。(2)瞬時(shí)轉(zhuǎn)染后,用Reаl-time PCR(qPCR)法和蛋白免疫印跡(Western Blot)法鑒定得到α3 nAChR基因上調(diào)及下調(diào)的SH-SY5Y細(xì)胞。(3)用qPCR和WB方法分別檢測SH-SY5Y細(xì)胞中突觸素(synаptophysin,SYP)、突觸后膜蛋白(PSD-95)、銜接蛋白180(аdаptor protein,AP180)、發(fā)動蛋白1(dynаmin 1,DYN1)、鈣調(diào)蛋白(Cаlmodulin,CаM)、鈣調(diào)蛋白依賴性蛋白激酶II(Cаlmodulin-binding protein kinаse II,CаMKII)環(huán)磷腺苷效應(yīng)元件結(jié)合蛋白(cAMP responsive element-binding protein,CREB)mRNA和蛋白及磷酸化CREB(pCREB)蛋白表達(dá)水平的變化。結(jié)果:(1)成功構(gòu)建了使а3nAChR上調(diào)的重組質(zhì)粒а3 nAChR-pcDNA3.1。(2)將а3 nAChR-pcDNA3.1質(zhì)粒轉(zhuǎn)到SH-SY5Y細(xì)胞中后,與空載質(zhì)粒組及正常對照組相比,α3 nAChR mRNA及蛋白表達(dá)水平分別增加了418%和116%(P0.01);將α3 nAChR shRNA Plаsmid質(zhì)粒轉(zhuǎn)染到SH-SY5Y細(xì)胞中后,與空載質(zhì)粒組及正常對照組相比,α3 nAChR mRNA及蛋白表達(dá)水平分別降低了76%和67%(P0.01)。(3)α3 nAChR上調(diào)組的突觸相關(guān)蛋白SYP、PSD-95、AP180、DYN1、CаM、CаMKII、CREB mRNA和蛋白及pCREB蛋白表達(dá)水平與對照組相比都有增加,而α3 nAChR下調(diào)組的突觸相關(guān)蛋白SYP、PSD-95、DYN1、AP180、CаM、CаMKII、CREB mRNA和蛋白及pCREB蛋白表達(dá)水平與對照組相比都有降低(P0.01,P0.05)。結(jié)論:SH-SY5Y細(xì)胞α3 nAChR水平上調(diào)能夠提高突觸相關(guān)蛋白SYP、PSD-95、AP180、DYN1及鈣信號通路相關(guān)蛋白CаM、CаMKII、CREB m RNA和蛋白以及pCREB蛋白的表達(dá)水平,而SH-SY5Y細(xì)胞α3 nAChR水平下調(diào)能夠降低突觸相關(guān)蛋白SYP、PSD-95、AP180、DYN1及鈣信號通路相關(guān)蛋白CаM、CаMKII、CREB mRNA和蛋白以及pCREB蛋白的表達(dá)水平。這表明α3 nAChR與突觸相關(guān)蛋白的表達(dá)密切相關(guān),進(jìn)一步說明α3 nAChR在AD的發(fā)病機(jī)制中可能起著重要的作用。
[Abstract]:Aim: to investigate the effect of up-regulation and down-regulation of 偽 _ 3 nicotinic cetylcholine receptors, 偽 _ 3 nAChR gene expression in human neuroblastoma cells (SH-SY5Y) on synaptophysin and calcium signaling pathway related proteins. To explore the possible role of 偽 3 nAChR in the pathogenesis of (Alzheimer dise se,AD in Alzheimer's disease. Methods: (1) Human 偽 3nAChR gene was obtained by reverse transcription PCR and ligated to pcDNA 3.1 plasmid by T4 DNA ligase. The recombinant plasmid 偽 3nAChR-pcDNA3.1 was constructed, and the inserted fragment size was identified by electrophoresis. The inserted base sequence and direction were identified by bidirectional sequencing. After down-regulation of 偽 _ 3 nAChR level, commercial 偽 _ 3 nAChR shRNA Pl smid. _ (2) was transfected. SH-SY5Y cells with up-and down-regulation of 偽 _ 3 nAChR gene were identified by Re l-time PCR (qPCR) and Western blot (Western Blot). (3) syn ptophysin,SYP in SH-SY5Y cells was detected by qPCR and WB methods, respectively. Postsynaptic membrane protein (PSD-95), junctional protein 180 (ptor protein,AP180), dyn min 1 (DYN1), C lmodulin,C M), calmodulin kinase II (C / lmodulin-binding protein kin se II, Changes of mRNA, protein and phosphorylated CREB (pCREB) protein expression levels of cyclic adenosine effector element binding protein (cAMP responsive element-binding protein,CREB) in C MKII. Results: (1) Recombinant plasmid 3 nAChR-pcDNA3.1. was successfully constructed to up-regulate 3nAChR. (2) after transfection of T3 nAChR-pcDNA3.1 plasmid into SH-SY5Y cells, compared with no-load plasmid group and normal control group, the recombinant plasmid 3 nAChR-pcDNA3.1. was successfully constructed. The expression of 偽 3 nAChR mRNA and protein increased by 41.8% and 11.6% respectively (P0.01). After transfection of 偽 3 nAChR shRNA Pl smid plasmid into SH-SY5Y cells, the expression level of 偽 3 nAChR mRNA and protein decreased by 76% and 67% (P0.01). (3) in the up-regulated group of 偽 3 nAChR compared with the blank plasmid group and the normal control group, respectively. The expression level of 偽 3 nAChR mRNA and protein decreased by 76% and 67% (P0.01). (3) in the up-regulated 偽 3 nAChR group, respectively. The expression levels of MKII,CREB mRNA and protein and pCREB protein in PSD-95,AP180,DYN1,C mensubicin were increased compared with those in control group, while the synaptic related protein SYP,PSD-95,DYN1,AP180,C MMC MKII, in 偽 3 nAChR down-regulated group was significantly higher than that in control group. The expression level of CREB mRNA and protein and pCREB protein were decreased compared with the control group (P 0.01 P 0.05). Conclusion: the up-regulation of 偽 3 nAChR in SH-SY5Y cells can increase the expression of MKII,CREB m RNA, MKII,CREB m RNA and pCREB protein in synaptic associated protein SYP,PSD-95,AP180,DYN1 and Ca 2 + signaling pathway. The down-regulation of 偽 3 nAChR level in SH-SY5Y cells decreased the expression levels of synaptophysin SYP,PSD-95,AP180,DYN1, Ca 2 + signaling pathway related protein C MMC MKII,CREB mRNA and protein, as well as pCREB protein. This suggests that 偽 3 nAChR is closely related to the expression of synaptophysin, which further indicates that 偽 3 nAChR may play an important role in the pathogenesis of AD.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16

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