核酶切割per基因所致的缓解吗啡成瘾作用研究  

The Effect of Alleviating Morphine Addiction Induced by Ribozyme which Specifically Cleaves per mRNA

作　　者：何畅[1] 屈艺[1] 尹华虎[1] 王若菡[2] 王正荣[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物医学工程研究室,成都610041 [2]四川大学华西基础医与法医学院重组DNA研究室

出　　处：《华西医科大学学报》2002年第4期493-496,共4页Journal of West China University of Medical Sciences

基　　金：国家自然科学基金 (批准号 3 0 0 70 2 78); CMB基金资助

摘　　要：目的　探讨 per基因与阿片类成瘾的相关性 ,进而寻找治疗阿片成瘾的基因戒毒疗法。方法　设计并合成特异性 per锤头状核酶基因和 per核酶靶 m RNA组分基因 ,并分别重组入体外转录质粒 ,而后将经体外转录反应得到 per核酶 RNA和地高辛标记的 per核酶靶 m RNA组分。将转录产物混合 ,在不同条件下进行体外切割反应后 ,采用地高辛化学发光法检测 per核酶的体外切割效率。小鼠脑室注射直接导入脂质体包裹的重组质粒 pc DNA3.1- per RZ DNA,按剂量递增法建立吗啡依赖动物模型 ,用纳洛酮催瘾 ,观察戒断反应的变化。结果per核酶对 per核酶靶 m RNA组分的体外切割效率达到 6 0 %。重组质粒组小鼠的刻板跳跃、“湿狗”样抖动和打洞次数与空质粒组、生理盐水组相比明显减少 ,但并不抑制小鼠的体重下降。结论　 per核酶具有体外定点切割 per基因 m RNA组分的活性。经小鼠吗啡成瘾模型检测 ,该重组质粒能成功转染活体小鼠脑细胞并在脑组织内稳定表达 per核酶 ,发挥阻断 per基因表达的作用 ,有效地减轻吗啡成瘾的戒断症状。Objective To investigate the role of period gene ( per) in the physical and psychological dependence of opiate with ribozyme technology in order to find a novel gene therapy approach for opiate addiction. Methods The hammerhead ribozyme DNA sequence toward per gene and the DNA sequence of per ribozyme's target RNA was devised and synthesized. Then, we introduced these two DNA sequences into plasmid vectors and transcribed them in vitro respectively. The transcripts of per ribozyme and its target DNA sequence were mixed followed by a specific ribozyme cleaving reaction to target per RNA. The recombinant plasmid containing per ribozyme DNA was transformed into the brains of mice in vivo. These mice were used to establish morphine dependent models. The precipitated withdrawal syndromes were induced by naloxone. Results The target per RNA was cleaved by per ribozyme at the site between 2187 and 2188, demonstrating 60% cleavage efficiency in vitro. The expression of per ribozyme in the brains of mice inhibited most morphine withdrawal syndromes such as the stereotype jumping behavior,'Wet dog'shaking, and burrowing ( P <0.01, compared with controls). However, the lost of body weight in mice was not alleviated. Conclusion The per ribozyme has specific cleavage activity toward per gene in vitro. The recombinant plasmid containing per ribozyme DNA is proved to transfect the brain cells of mice successfully, with stable expression of per ribozyme, which blocks per expression and thus alleviats morphine withdrawal syndromes.

关 键 词：阿片成瘾 核酶体外切割 period基因 吗啡 

分 类 号：R749.61[医药卫生—神经病学与精神病学]