选择性下调大鼠ACC神经元NR2B受体表达的小RNA干扰慢病毒载体的构建

发布时间：2018-05-20 16:06

本文选题：前扣带皮层 + 恶性情绪体验　；参考：《山东大学》2010年硕士论文

【摘要】： 研究背景疼痛是一种与组织损伤或潜在损伤相关的不愉快的主观感觉和情绪体验。该定义赋予疼痛两层含义：感觉分辨和情绪体验。前者主要编码痛刺激的属性,包括痛刺激的性质,强度和位置等；后者编码痛刺激引起的厌恶,焦虑,恐惧等负面情绪。临床观察表明,慢性痛如晚期癌痛患者遭受的恶性情绪体验,如焦虑,恐惧,孤独甚至厌世等给病人造成的身心伤害远比疾病本身更为严重。因此,缓解和治疗慢性痛病人的恶性情绪体验,对治疗疾病,改善疼痛病人的生活质量具有重要意义。大量研究表明,大脑边缘系统的前扣带皮层(Anterior Cingulate Cortex,ACC)在慢性痛恶性情绪体验的形成过程中起着重要作用。N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受体是一种配体门控离子型谷氨酸受体参与体内神经发育,突触可树性,学习记忆以及痛觉信号的转导等生理病理过程。已经证实,ACC神经元NMDA受体在恶性情绪体验形成中起着重要作用。NMDA受体根据构成亚基的不同分为2A,2B,2C和2D四种,而ACC神经元以2B为主,即NR2B。研究目的为进一步研究NR2B受体在恶性情绪体验形成中的作用,本实验拟通过小RNA干扰技术,制作可选择性下调大鼠前扣带皮层神经元NR2B基因表达的慢病毒载体。研究方法1.NR2B基因特异性的siRNA靶序列的设计与合成：根据siRNA的设计原则,在GenBank中查找大鼠的ACC神经元NR2B的mRNA全序列(GeneBank号为NM_012574),通过Blast软件确定与其它非相关基因无同源性,按照pFU-GW-iRNA载体位点的要求设计3对寡核苷酸。 2. NR2B/siRNA重组慢病毒载体的构建：根据筛选的NR2B/siRNA序列,设计并合成两条可退火产生短双链的引物,经过变性,复性后形成两端带有HpaI和Xhol位点的双链寡核苷酸片段。pFU-GW-iRNA质粒是一种常用的RNAi表达质粒,该质粒含有CMV和U6启动子,分别启动EGFP和目的siRNA的表达。选取U6启动子后MCS区Hpa 1和Xhol位点连入上述合成的双链寡核苷酸片段,PCR鉴定,获取重组质粒。 3.最佳干扰靶点的筛选：构建好含有目的基因的表达克隆质粒和针对靶基因不同干扰靶点的RNAi病毒载体质粒,根据Invitrogen公司的lipofectamine2000使用说明共转染培养好的工具细胞(即293T细胞),转染24h荧光显微镜下观测转染效果,转染36h收集细胞抽提蛋白,采用Western blot检测目的蛋白的表达情况,进而判断不同靶点的干扰效果。结果通过酶切和测序证实NR2B/siRNA序列正确插入pFU-GW-iRNA载体,成功构建了三种含NR2B/siRNA序列的慢病毒载体,分别为NR2B/siRNA慢病毒载体Ⅰ、Ⅱ和Ⅲ。将合成的三种NR2B/siRNA漫病毒载体与NR2B表达质粒共转染293T细胞并以Westernblot检测NR2B的表达,结果证实三种慢病毒载体对目的基因均有敲减作用,其中以NR2B/siRNA'慢病毒载体Ⅲ敲减效果最佳。结论成功建立可选择性下调大鼠ACC神经元NR2B基因的小干扰RNA慢病毒载体,为进一步研究奠定了基础。
[Abstract]:Background pain is an unpleasant subjective feeling and emotional experience associated with tissue damage or potential damage. The definition gives two meanings to pain: sensory and emotional experience. The former mainly encodes the properties of pain stimuli, including the nature, intensity and location of pain stimuli, and the latter encodes aversion and anxiety caused by pain stimulation. Clinical observation shows that the malignant emotional experience of patients with chronic pain such as advanced cancer pain, such as anxiety, fear, loneliness and even the world weariness, is much more serious than the disease itself. Therefore, the malignant emotional experience of the patients with chronic pain, the treatment of the disease, the improvement of the pain of the patient's life, and the treatment of the chronic pain. The quality of living is of great significance.
A large number of studies have shown that the Anterior Cingulate Cortex (ACC) in the cerebral marginal system plays an important role in the formation of chronic painful emotional experience,.N- methyl -D- aspartic acid (N-methyl-D-aspartate, NMDA) receptor is a ligand gated ionotropic glutamate receptor involved in the body's neurodevelopment, synaptic tree, and learning. It has been proved that the NMDA receptor in ACC neurons plays an important role in the formation of malignant emotional experience. The.NMDA receptor is divided into four types, 2A, 2B, 2C and 2D, and ACC neurons are dominated by 2B, that is NR2B..
The purpose of this study is to further study the role of NR2B receptor in the formation of malignant emotional experience. This experiment is intended to produce a lentivirus vector that can selectively downregulate the expression of NR2B gene in the neurons of the anterior cingulate cortex by small RNA interference.
The design and synthesis of the specific siRNA target sequence of 1.NR2B gene: according to the design principle of siRNA, the mRNA full sequence of NR2B in ACC neurons of rats was found in GenBank (GeneBank No. NM_012574), and the homology of the other non related genes was determined by Blast software, and 3 pairs of oligonucleotides were designed according to the requirement of the pFU-GW-iRNA carrier position. Glucoside acid.
2. NR2B/siRNA recombinant lentivirus vector construction: designed and synthesized two annealed short double stranded primers based on the screened NR2B/siRNA sequence. After denaturation, the double stranded oligonucleotide fragment.PFU-GW-iRNA plasmid with HpaI and Xhol loci at both ends is a common RNAi expression plasmid, which contains CMV and U6 promoter. The expression of EGFP and target siRNA was started respectively. The Hpa 1 and Xhol loci of the MCS region after U6 promoter were linked into the synthesized double stranded oligonucleotide fragment, and the recombinant plasmid was obtained by PCR identification.
3. the selection of the best target of interference: construct a good expression plasmid containing the target gene and RNAi vector plasmid with different targets for target gene, CO transfection the good tool cells (i.e. 293T cells) according to the lipofectamine2000 usage of Invitrogen company, observe the transfection effect under the 24h fluorescence microscope, and transfect the transfection 36 H collected cell extract protein and detected the expression of target protein by Western blot, and then judged the interference effect of different targets.
Results the NR2B/siRNA sequence was correctly inserted into pFU-GW-iRNA vector by enzyme digestion and sequencing, and three kinds of lentivirus vectors containing NR2B/siRNA sequences were successfully constructed, which were NR2B/siRNA lentivirus vector I, II and III respectively. The three NR2B/siRNA diffuse vectors were co transfected with NR2B expression plasmid and 293T cells were co transfected and NR2B was detected with Westernblot. The results showed that three lentiviral vectors had knock down effects on target genes, and the lentiviral vector NR2B/siRNA'was the best knockdown effect.
Conclusion the small interfering RNA lentiviral vector which can selectively downregulate the NR2B gene of rat ACC neurons has been successfully established, which lays the foundation for further research.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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