人神经元性AADC基因的克隆、测序及其对帕金森病的治疗作用

背景帕金森病患者脑内黑质多巴胺能神经元变性导致芳香族氨基酸脱羧酶(aromaticL-amino acid decarboxylase,AADC)活性水平的降低及波动是产生帕金森患者临床症状和在L-dopa替代治疗时"症状波动"等不良反应出现的重要原因之一.目的探讨AADC基因脑内移植对帕金森病的治疗作用及其在左旋多巴治疗过程中的地位,并探讨阳离子脂质体介导的基因治疗方法的有效性.设计2×2析因设计.地点和材料中山大学附属第二医院林百欣医学研究中心,材料主要包括人嗜铬细胞瘤组织,TRIzol,真核表达载体质粒pCDNA3,阳离子脂质体,6-OHDA,阿朴吗啡,AADC单克隆抗体,SD大鼠.干预使用基因克隆、亚克隆技术构建pCDNA3-AADC真核表达载体;将帕金森病模型的SD大鼠随机分为实验组与对照组,分别以pCDNA3-AADC重组体和pCDNA3空载体进行阳离子脂质体包裹后,注射于帕金森病SD大鼠纹状体内,并在该基础上对两组大鼠分别采用一定浓度的L-dopa进行治疗.采用免疫组化方法确定AADC的表达.主要观察指标①AADC基因脑内移植后两组大鼠的旋转行为.②采用一定浓度的L-dopa进行治疗对大鼠旋转行为的影响.结果①阳离子脂质体包裹的pCDNA3-AADC重组体脑内注射后,在3,7,14,21,28d时大鼠的旋转行为获得了一定的改善(P＜0.05),分别改善了56.0%,62.6%,49.4%,36.3%,27.4%,以1周时最为明显,在第5周时,其旋转行为则于移植前无明显差异(P＞0.05).②使用同等浓度的左旋多巴(L-dopa,10mg/kg体质量·d)治疗后,在上述相同的时间点时,实验组较对照组明显改善模型大鼠的旋转行为(P＜0.05),以1周时最为明显,其旋转行为改善达93.4%,在第5周时,两组间差异无显著意义(P＞0.05).③免疫组化方法确定AADC在纹状体区的稳定表达.结论增加脑内AADC基因表达可有效改善帕金森病大鼠的旋转行为,并可增加L-dopa治疗效果,有助在低剂量上长期维持L-dopa的疗效;阳离子脂质体介导的裸基因脑内移植技术为基因治疗帕金森病提供一种手段.

Cloning and sequencingof human neuronal aromatic L-amino acid decarboxylase gene and its therapeutic effects onParkinson''''s disease

BACKGROUND: In Parkinson's disease, the activity of aromatic L-amino acid decarborJase (AADC) decreased greatly secondary to the degeneration of dopaminergic neurons within the substantia nigra pars compacta leads to a series of clinical symptoms occur. Furthermore, the fluctuation of AADC activity is one of the most important cause leading to some severe side-effects,such as "on-off" phenomenon and so on, following the chronic L-dopa administration.OBJECTIVE: To study the roles of AADC gene therapy for Parkinson' s disease and its action by coadministrated of exogenous L-dopa and to discuss the effectiveness of the approach of cationic liposome mediated gene transferring into the brain.DESIGN: 2 x 2 factorial design.SETTING and MATERIALS: Research was completed at Linbaixin Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University. Materials used mainly includes human pheochromocytoma, TRIzoleukaryotic expression vector pCDNA3, cationic liposome, 6-OHDA, apomorphine, AADC monoclone antibody, SD rats.INTERVENTIONS: pCDNA3-AADC complex were constructed using gene cloning and subcloning technics. Then either the pCDNA3-AADC-Liposome or pCDNA3-Liposome complexes were injected into the striatum of Parkinsonian SD rats of experimental and control groups respectively. Furthermore,exogenous L-dopa were given to each groups. The expression of AADC were assayed with immunohistochemical staining.therapy at a certain concentration on rotational behaviour of rats.complex improved the rotational behavior of Parkinsonian rats in some degree when its being observed at 3, 7, 14, 21 and 28 days( P ＜ 0.05), reached56.0%, 62.6%, 49.4% , 36. 3%, 27.4%, respectively, this improvement lasted for 4 weeks and disappeared at the 5th week, the maximum improveexogenous L-dopa( 10 mg/kg body mass) per day, the experimental groups reached much more obvious improvement than that in the controls at the same time points( P ＜ 0.05), the maximum improvement was also at the time of 1munohistochemical staining showed that the AADC were expressed stably in the striatum.CONCLUSION: Increase of AADC gene expression may improve the rotational behavior of Parkinsonian rats as well as elevate the curative effect of exogenous L-dopa therapy, suggesting that it should be helpful to maintain a long-term L-dopa theraputic effect at a much lower level. The technic of intracerebral gene transfer mediated by cationic liposome may be a new method for gene therapy of PD.