SH-SY5Y细胞α3尼古丁受体表达水平的改变对突触相关蛋白的影响

发布时间：2019-02-17 09:06

【摘要】：目的:研究人神经母细胞瘤细胞(SH-SY5Y)α3尼古丁受体(а3 nicotinic аcetylcholine receptors,α3 nAChR)基因表达上调及下调后对细胞突触相关蛋白及钙信号通路相关蛋白的影响,从而探讨α3 nAChR在阿尔茨海默病(Alzheimer diseаse,AD)发病机制中的可能作用。方法:(1)采用逆转录PCR的方法获取人α3nAChR基因,用T4 DNA连接酶将其连接到pcDNA 3.1质粒,构建重组质粒α3nAChR-pcDNA3.1,电泳鉴定插入片段大小,双向测序鉴定插入碱基序列和方向;下调α3 nAChR水平选择商品化的α3 nAChR shRNA Plаsmid。(2)瞬时转染后,用Reаl-time PCR(qPCR)法和蛋白免疫印迹(Western Blot)法鉴定得到α3 nAChR基因上调及下调的SH-SY5Y细胞。(3)用qPCR和WB方法分别检测SH-SY5Y细胞中突触素(synаptophysin,SYP)、突触后膜蛋白(PSD-95)、衔接蛋白180(аdаptor protein,AP180)、发动蛋白1(dynаmin 1,DYN1)、钙调蛋白(Cаlmodulin,CаM)、钙调蛋白依赖性蛋白激酶II(Cаlmodulin-binding protein kinаse II,CаMKII)环磷腺苷效应元件结合蛋白(cAMP responsive element-binding protein,CREB)mRNA和蛋白及磷酸化CREB(pCREB)蛋白表达水平的变化。结果:(1)成功构建了使а3nAChR上调的重组质粒а3 nAChR-pcDNA3.1。(2)将а3 nAChR-pcDNA3.1质粒转到SH-SY5Y细胞中后,与空载质粒组及正常对照组相比,α3 nAChR mRNA及蛋白表达水平分别增加了418%和116%(P0.01);将α3 nAChR shRNA Plаsmid质粒转染到SH-SY5Y细胞中后,与空载质粒组及正常对照组相比,α3 nAChR mRNA及蛋白表达水平分别降低了76%和67%(P0.01)。(3)α3 nAChR上调组的突触相关蛋白SYP、PSD-95、AP180、DYN1、CаM、CаMKII、CREB mRNA和蛋白及pCREB蛋白表达水平与对照组相比都有增加,而α3 nAChR下调组的突触相关蛋白SYP、PSD-95、DYN1、AP180、CаM、CаMKII、CREB mRNA和蛋白及pCREB蛋白表达水平与对照组相比都有降低(P0.01,P0.05)。结论:SH-SY5Y细胞α3 nAChR水平上调能够提高突触相关蛋白SYP、PSD-95、AP180、DYN1及钙信号通路相关蛋白CаM、CаMKII、CREB m RNA和蛋白以及pCREB蛋白的表达水平,而SH-SY5Y细胞α3 nAChR水平下调能够降低突触相关蛋白SYP、PSD-95、AP180、DYN1及钙信号通路相关蛋白CаM、CаMKII、CREB mRNA和蛋白以及pCREB蛋白的表达水平。这表明α3 nAChR与突触相关蛋白的表达密切相关,进一步说明α3 nAChR在AD的发病机制中可能起着重要的作用。
[Abstract]:Aim: to investigate the effect of up-regulation and down-regulation of 伪 _ 3 nicotinic cetylcholine receptors, 伪 _ 3 nAChR gene expression in human neuroblastoma cells (SH-SY5Y) on synaptophysin and calcium signaling pathway related proteins. To explore the possible role of 伪 3 nAChR in the pathogenesis of (Alzheimer dise se,AD in Alzheimer's disease. Methods: (1) Human 伪 3nAChR gene was obtained by reverse transcription PCR and ligated to pcDNA 3.1 plasmid by T4 DNA ligase. The recombinant plasmid 伪 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 伪 _ 3 nAChR level, commercial 伪 _ 3 nAChR shRNA Pl smid. _ (2) was transfected. SH-SY5Y cells with up-and down-regulation of 伪 _ 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 伪 3 nAChR mRNA and protein increased by 41.8% and 11.6% respectively (P0.01). After transfection of 伪 3 nAChR shRNA Pl smid plasmid into SH-SY5Y cells, the expression level of 伪 3 nAChR mRNA and protein decreased by 76% and 67% (P0.01). (3) in the up-regulated group of 伪 3 nAChR compared with the blank plasmid group and the normal control group, respectively. The expression level of 伪 3 nAChR mRNA and protein decreased by 76% and 67% (P0.01). (3) in the up-regulated 伪 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 伪 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 伪 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 伪 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 伪 3 nAChR is closely related to the expression of synaptophysin, which further indicates that 伪 3 nAChR may play an important role in the pathogenesis of AD.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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