發(fā)布時(shí)間：2018-03-25 05:09

  本文選題：前腦啡肽原基因　切入點(diǎn)：逆轉(zhuǎn)錄病毒　出處：《鄭州大學(xué)》2006年碩士論文

【摘要】：慢性疼痛，尤其是癌性疼痛和頑固性疼痛一直是醫(yī)療界的一道難題。傳統(tǒng)的鎮(zhèn)痛藥尚不能有效治療慢性疼痛尤其癌痛。腦啡肽作為一種內(nèi)源性阿片肽類鎮(zhèn)痛藥受到關(guān)注，用于許多動(dòng)物實(shí)驗(yàn)已證明有明顯的鎮(zhèn)痛效應(yīng)。將前腦啡肽原基因做為目的基因，利用逆病毒載體，經(jīng)過包裝細(xì)胞系的包裝，產(chǎn)生的病毒可將前腦啡肽原基因整合到靶細(xì)胞NIH3T3細(xì)胞的基因組中，制造出可以分泌鎮(zhèn)痛物質(zhì)腦啡肽的工程化細(xì)胞系，利用人工大量獲得。本實(shí)驗(yàn)采用pLNCX2載體，其中5′LTR(包括病毒啟動(dòng)子／增強(qiáng)序列)可控制neo~r基因的表達(dá)，用于真核細(xì)胞中抗生素篩選。目的基因可克隆進(jìn)多克隆位點(diǎn)，在P_(CMVIE)控制下表達(dá)。pLNCX_2含有包裝信號(hào)Ψ序列，但沒有g(shù)ag、pol和env等編碼病毒結(jié)構(gòu)蛋白的基因，須通過包裝細(xì)胞系(PT67等)的包裝。因此，當(dāng)轉(zhuǎn)染細(xì)胞增殖傳代時(shí)，其形成復(fù)制完整病毒能力的的機(jī)會(huì)極少。其包裝產(chǎn)生的病毒，攜帶前腦啡肽源基因，，可作為疼痛基因治療的實(shí)驗(yàn)研究的有力工具。 目的 研究利用RT-PCR技術(shù)，可否獲得人前腦啡肽原基因，并采用pLNCX2逆轉(zhuǎn)錄病毒載體，將外源性腦啡肽基因?qū)氩⒄系桨屑?xì)胞NIH3T3細(xì)胞的基因組中，觀察是否有外源性腦啡肽基因的表達(dá)，探討一條有效的治療疼痛的轉(zhuǎn)基因方法。 方法 1．RT-PCR 根據(jù)GenBank報(bào)道的人腦啡肽基因序列(NM_006211)，設(shè)計(jì)引物，其引物5’與3’端分別加上HindⅢ和NotⅠ酶切位點(diǎn)，以便目的基因插入逆轉(zhuǎn)錄病毒載體。提取人腦組織總RNA，然后反轉(zhuǎn)錄得到包含前腦啡肽原基因的單鏈總cDNA，
[Abstract]:Chronic pain, especially cancerous pain and intractable pain, has been a difficult problem in the medical field. Traditional analgesics have not been effective in the treatment of chronic pain, especially cancer pain. Enkephalin has attracted much attention as an endogenous opioid peptide analgesics. The proenkephalin gene was used as the target gene in many animal experiments. The virus can integrate the proenkephalin gene into the genome of the target cell NIH3T3 cells and produce an engineering cell line which can secrete the analgesic substance enkephalin. The expression of neo~r gene (including virus promoter / enhancer sequence) can be controlled by 5 LTRs for antibiotic screening in eukaryotic cells. The target gene can be cloned into polyclonal sites and expressed under the control of Pappa CMVIE.pLNCX2 contains the package signal 蠄 sequence. But without genes that encode viral structural proteins such as Gagapol and env, they have to be packaged through a packaging cell line, such as PT67, etc. Therefore, when transfected cells proliferate, they have little chance of forming the ability to replicate complete viruses. Carrying proenkephalin gene can be used as a powerful tool for experimental study of pain gene therapy. Purpose. To study whether human proenkephalin gene can be obtained by using RT-PCR technique, and to introduce exogenous enkephalin gene into the genome of target cell NIH3T3 cells by pLNCX2 retrovirus vector, and to observe whether there is exogenous enkephalin gene expression. To explore an effective transgenic method for the treatment of pain. Method. 1.RT-PCR. According to the sequence of human brain enkephalin gene reported by GenBank, NM006211A was designed and primers were designed. The primers 5 'and 3' end of the primer were digested with Hind 鈪

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