BACKGROUND: Heroin, which is characterized by strong liposolubility and immediate effect, can rapidly enter central nervous system through blood brain barrier; however, mechanism of its dependence is still unclear up to now. Therefore, it becomes a hot topic to investigate mechanism of molecular biology of drug dependence, especially to research changes of gene expression.OBJECTIVE: To find out the expression of specific gene related to heroin dependence so as to elucidate the mechanism of molecular biology of heroin dependence.DESIGN: Randomized controlled observation on the basis of experimental animals.SETTING: Center of Psychohygiene, the First Hospital affiliated to Chongqing Medical University.MATERIALS: Eight C57BL/6J mice were randomly divided into experimental group and control group with 4 in each group. Drug and equipments: heroin, Tripure separating agent, PolyATtract mRNA separating system Ⅲ, etc.METHODS: Heroin-dependent models in mice were established by dose-increasing hypodermical injection of heroin for 7 days. On the 7th day,80 mg/kg heroin was injected once, and 2 hours later, 5 mg/kg naloxone was injected'. Mice in control group were injected with the same volume of saline and the same dosage of naloxone. Then, mice were put in glass cage to observe jumping times and numbers of animals which had ptosis, diarrhea, wet-dog movement and trembling of anterior claws within 15 minutes.Mice were immediately sacrificed and total RNA and mRNA were extracted from the brains in experimental and control groups with Trripure separating kit, and then, suppressive subtractive hybridization was proceeded.The hybridization sample was amplified by PCR and analyzed with gel electrophoresis.MAIN OUTCOME MEASURES: Quantity of total RNA and mRNA in brain tissue and different expression of cDNA sections in brain.RESULTS: Mice in each group were involved in the final analysis. ①Results of abstinent symptom after injection of naloxone: Abstinent symptom was observed on mice in experimental group. Jumping times within 15 minutes were 9.75±1.65, which was significant difference from those in control group (P ＜ 0.01). ② Comparison of total RNA and mRNA in brain tissue and different expression of cDNA sections in brain: Quantities of total RNA and mRNA were higher in experimental group than those in control group, and difference of mRNA was significant (P ＜ 0.05). After the second suppressive subtractive hybridization, mixture was amplified with PCR and nest-like PCR to differently express cDNA sections.CONCLUSION: Withdrawal of heroin dependence may cause changes of gene expression, and there is probably an expression of the specific gene in the brain of heroin-dependent mice.%背景:海洛因能很快透过血脑屏障进入中枢神经系统,其脂溶性强,起效快,然而药物依赖的发生机制至今尚未完全明了,探索药物依赖的分子生物学机制,特别是基因表达改变的研究已成为该领域研究的热点.目的:探索与海洛因依赖性形成有关的特异性基因表达,以期阐明海洛因依赖的分子生物学机制.设计:以实验动物为观察对象的随机对照观察.单位:重庆医科大学附属第一医院心理卫生中心.材料:C57BL/6J小鼠8只,随机分为实验组和对照组,每组各4只.药品和试剂有海洛因、Tripure分离试剂、PolyATtract m RNA分离系统Ⅲ等.方法:实验组小鼠采用递增法皮下注射海洛因法建立海洛因依赖小鼠模型,连续注射7天,第7天9:00一次皮下注射80mg/kg,2 h后腹腔注射纳洛酮5 mg/kg.对照组小鼠皮下注射同体积的生理盐水及同剂量的纳洛酮.注射纳洛酮后立即将各组小鼠放入玻璃观察罩内,观察15 min内跳跃次数,15 min内出现眼睑下垂、腹泻、湿狗样拌动及前爪震颤的动物数,之后马上断头处死,采用Trripure分离试剂盒分别提取实验组和对照组脑组织的总RNA及其全部mRNA.然后进行抑制性消减杂交,杂交产物扩增,凝胶电泳分析.主要观察指标:实验组和对照组脑组织总的RNA、mRNA量;海洛因依赖小鼠脑内差异表达的cDNA片断.结果:各组小鼠测试结果全部进入结果分析.①注射纳洛酮后各组戒断症状观察结果:实验组小鼠腹腔注射纳洛酮后皆出现戒断症状,15 min内跳跃次数为(9.75±1.65)次,与对照组比较差异有显著性(P＜0.01).②两组小鼠脑组织总RNA、mRNA量的比较及海洛因依赖小鼠脑内差异表达的cDNA片断:实验组小鼠脑内总RNA和mRNA量均较对照组高,mRNA的差异具有显著性(P＜0.05),经二轮轮抑性消减杂交后,获得杂交混合物,经PCR和巢式PCR扩增,获得了差异表达的cDNA片断.结论:海洛因依赖戒断可引起基因表达的改变,海洛因依赖小鼠脑内可能存在特异性基因表达.
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