發(fā)布時(shí)間：2018-05-20 16:06

  本文選題：前扣帶皮層 + 惡性情緒體驗(yàn)�。� 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 研究背景疼痛是一種與組織損傷或潛在損傷相關(guān)的不愉快的主觀感覺和情緒體驗(yàn)。該定義賦予疼痛兩層含義：感覺分辨和情緒體驗(yàn)。前者主要編碼痛刺激的屬性,包括痛刺激的性質(zhì),強(qiáng)度和位置等；后者編碼痛刺激引起的厭惡,焦慮,恐懼等負(fù)面情緒。臨床觀察表明,慢性痛如晚期癌痛患者遭受的惡性情緒體驗(yàn),如焦慮,恐懼,孤獨(dú)甚至厭世等給病人造成的身心傷害遠(yuǎn)比疾病本身更為嚴(yán)重。因此,緩解和治療慢性痛病人的惡性情緒體驗(yàn),對治療疾病,改善疼痛病人的生活質(zhì)量具有重要意義。 大量研究表明,大腦邊緣系統(tǒng)的前扣帶皮層(Anterior Cingulate Cortex,ACC)在慢性痛惡性情緒體驗(yàn)的形成過程中起著重要作用。N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受體是一種配體門控離子型谷氨酸受體參與體內(nèi)神經(jīng)發(fā)育,突觸可樹性,學(xué)習(xí)記憶以及痛覺信號(hào)的轉(zhuǎn)導(dǎo)等生理病理過程。已經(jīng)證實(shí),ACC神經(jīng)元NMDA受體在惡性情緒體驗(yàn)形成中起著重要作用。NMDA受體根據(jù)構(gòu)成亞基的不同分為2A,2B,2C和2D四種,而ACC神經(jīng)元以2B為主,即NR2B。 研究目的為進(jìn)一步研究NR2B受體在惡性情緒體驗(yàn)形成中的作用,本實(shí)驗(yàn)擬通過小RNA干擾技術(shù),制作可選擇性下調(diào)大鼠前扣帶皮層神經(jīng)元NR2B基因表達(dá)的慢病毒載體。 研究方法1.NR2B基因特異性的siRNA靶序列的設(shè)計(jì)與合成：根據(jù)siRNA的設(shè)計(jì)原則,在GenBank中查找大鼠的ACC神經(jīng)元NR2B的mRNA全序列(GeneBank號(hào)為NM_012574),通過Blast軟件確定與其它非相關(guān)基因無同源性,按照pFU-GW-iRNA載體位點(diǎn)的要求設(shè)計(jì)3對寡核苷酸。 2. NR2B/siRNA重組慢病毒載體的構(gòu)建：根據(jù)篩選的NR2B/siRNA序列,設(shè)計(jì)并合成兩條可退火產(chǎn)生短雙鏈的引物,經(jīng)過變性,復(fù)性后形成兩端帶有HpaI和Xhol位點(diǎn)的雙鏈寡核苷酸片段。pFU-GW-iRNA質(zhì)粒是一種常用的RNAi表達(dá)質(zhì)粒,該質(zhì)粒含有CMV和U6啟動(dòng)子,分別啟動(dòng)EGFP和目的siRNA的表達(dá)。選取U6啟動(dòng)子后MCS區(qū)Hpa 1和Xhol位點(diǎn)連入上述合成的雙鏈寡核苷酸片段,PCR鑒定,獲取重組質(zhì)粒。 3.最佳干擾靶點(diǎn)的篩選：構(gòu)建好含有目的基因的表達(dá)克隆質(zhì)粒和針對靶基因不同干擾靶點(diǎn)的RNAi病毒載體質(zhì)粒,根據(jù)Invitrogen公司的lipofectamine2000使用說明共轉(zhuǎn)染培養(yǎng)好的工具細(xì)胞(即293T細(xì)胞),轉(zhuǎn)染24h熒光顯微鏡下觀測轉(zhuǎn)染效果,轉(zhuǎn)染36h收集細(xì)胞抽提蛋白,采用Western blot檢測目的蛋白的表達(dá)情況,進(jìn)而判斷不同靶點(diǎn)的干擾效果。 結(jié)果通過酶切和測序證實(shí)NR2B/siRNA序列正確插入pFU-GW-iRNA載體,成功構(gòu)建了三種含NR2B/siRNA序列的慢病毒載體,分別為NR2B/siRNA慢病毒載體Ⅰ、Ⅱ和Ⅲ。將合成的三種NR2B/siRNA漫病毒載體與NR2B表達(dá)質(zhì)粒共轉(zhuǎn)染293T細(xì)胞并以Westernblot檢測NR2B的表達(dá),結(jié)果證實(shí)三種慢病毒載體對目的基因均有敲減作用,其中以NR2B/siRNA'慢病毒載體Ⅲ敲減效果最佳。 結(jié)論成功建立可選擇性下調(diào)大鼠ACC神經(jīng)元NR2B基因的小干擾RNA慢病毒載體,為進(jìn)一步研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

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