海马神经元转染沉默AP4M1基因的慢病毒后AMPA受体的表达变化

发布时间：2018-02-09 00:53

本文关键词： 海马神经元原代培养慢病毒 AP4M1 AMPA受体　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的原代培养胎鼠海马神经元,并转染包装了沉默衔接蛋白复合物-4μ亚基(adaptor-related protein complex 4,mu 1 subunit;AP4M1)基因的慢病毒后,观察细胞AP4M1、GluR1及GluR2(AMPA受体)的表达变化,以探究AP4M1可能介导的AMPA受体运输变化的分子机制。材料及方法本课题将原代培养海马神经作为实验对象,应用免疫荧光来标记神经元特异性烯醇化酶(neuron-specifice nolase,NSE),来鉴定所培养细胞中神经元的纯度。细胞培养中进行慢病毒(沉默AP4M1基因)转染,并进行荧光检测,以鉴定转染效率,实验分组:阴性慢病毒转染组及阳性慢病毒转染组(AP4M1基因表达沉默)。分别应用Real-time PCR和Western blotting方法研究AP4M1、GluR1及GluR2的mRNA及蛋白水平的表达变化。结果1、海马神经元的培养、鉴定及转染原代培养的SD大鼠胎鼠的海马神经细胞生长良好,在种植后第7天神经元基本发育成熟,神经元免疫荧光染色检测结果显示所培养细胞中的神经元纯度大于95%。在培养第2天可行慢病毒的转染,在转染8-12小时后换液,并于72小时后重复转染1次,再转染8-12h,72小时后进行荧光检测,转染率大于85%。2、海马神经元转染慢病毒后AP4M1、GluR1及GluR2的表达变化1)Real-time PCR检测:阳性病毒组AP4M1 mRNA表达量较阴性病毒组显著下调(t=68.193,P0.01,n=8);转染后GluR1、GluR2 mRNA表达量较阴性病毒组也明显下降(t=8.060,P0.01,n=8);(t=8.154,P0.01,n=8)。2)Western blot检测:转染阳性病毒后AP4M1、GluR1及GluR2蛋白表达量较阴性病毒组显著下调(F=7.566,t′=15.966,P0.01,n=8),(F=0.408,t′=8.690,P0.01,n=8)(F=5.560,t′=13.770,P0.01,n=8)。结论海马神经元在转染了包埋沉默AP4M1基因的慢病毒后,AP4M1的mRNA和蛋白表达明显下调,说明转染有效,进而海马神经元的GluR1、2的mRNA和蛋白表达也明显下调;AP4M1可能直接或间接调控海马神经元中的GluR1、2的转运。
[Abstract]:The purpose of primary cultured hippocampal neurons of fetal rats, and transfected the silence adaptor protein complex -4 subunit (adaptor-related protein complex 4, mu 1 subunit; AP4M1) lentivirus, AP4M1 and GluR1 were observed, GluR2 (AMPA receptor) expression and molecular mechanism of changes of AMPA receptor mediated transport to explore AP4M1. Materials and methods in this study, primary cultured hippocampal neurons as the experiment object, using immunofluorescence labeled neuron specific enolase (neuron-specifice nolase, NSE), to identify the cultured cells. The purity of neurons in cell culture (lentivirus silencing AP4M1 gene transfection), and fluorescence detection, to identify the transfection efficiency of lentivirus transfected experimental groups: negative group and positive group (transfected with lentiviral AP4M1 gene silencing). Real-time and Western were used to study the PCR blotting method AP4 M1, mRNA expression and protein level of GluR1 and GluR2. The results of 1 cultured hippocampal neurons, hippocampal neurons of SD fetal rat identification and transfection of primary culture grew well at seventh days after planting basically mature neurons, neurons were detected by immunofluorescence staining showed that the purity of cultured neuronal cells more than 95%. in transfected cultured second days feasible lentivirus was replaced, in 8-12 hours after transfection, and in 72 hours after transfection and then repeated 1 times, 72 hours after the transfection of 8-12h, fluorescence detection, transfection rate is more than 85%.2, hippocampal neurons transfected with lentiviral AP4M1 expression of GluR1 and GluR2 1 Real-time) detection of PCR virus group AP4M1: the positive expression of mRNA virus group significantly decreased compared to negative (t=68.193, P0.01, n=8); GluR1 after transfection, GluR2 mRNA expression was negative in virus group decreased significantly (t=8.060, P0.01, n=8 (t=8.154, P0.01), N=8).2 Western) blot detection: transfection positive virus AP4M1, significantly reduced the amount of virus group compared with the negative expression of GluR1 and GluR2 proteins (F=7.566, t '=15.966, P0.01, n=8), (F=0.408, t' =8.690, P0.01, n=8) (F=5.560, t '=13.770, P0.01, n=8). Conclusion the sea horse the embedding of AP4M1 gene silencing in neurons transfected with lentivirus, mRNA and protein expression of AP4M1 was significantly reduced, indicating effective transfection, and the expression of mRNA and protein in hippocampal neurons of GluR1,2 also significantly decreased; AP4M1 may directly or indirectly regulate hippocampal neurons in the translocation of GluR1,2.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742

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