HO-1基因转染对慢性脑缺血大鼠海马乙酰胆碱转移酶表达的影响

Effect of transfection of heine oxygenase-1 gene on choline acetyltransferase expression in hippocampus in rats with chronic cerebral ischemia

目的 探讨重组腺相关病毒介导血红素加氧酶-1（rAAV-rHO-1）基因转染对慢性脑缺血大鼠海马乙酰胆碱转移酶（ChAT）表达的影响。方法 健康雄性清洁级SD大鼠54只，体重250～300g，10～11月龄，随机分成3组（n=18）：假手术组（SH组）、慢性脑缺血组（IS组）和rAAV-rHO-1治疗组（HO-1组）。HO-1组大鼠海马内注射rAAV-rHO-12μl 1×10^9 v．g．，SH组和IS组注入等容量生理盐水。注射rAAV-rHO-1后7d时，采用永久结扎双侧颈总动脉的方法制备慢性脑缺血模型。大鼠慢性脑缺血2个月后进行Morris水迷宫试验，测定学习记忆功能，随后每组处死15只大鼠，采用免疫组织化学法测定海马CA1区ChAT表达，采用免疫荧光化学染色法测定海马CA1区HO-1的表达，光镜下观察海马CA1区病理学结果，并计数神经元；每组处死3只大鼠，电镜下观察海马CA1区超微结构，并计数突触。结果 与SH组比较，IS组学习记忆能力降低，海马CA1区神经元计数和突触计数减少，IS组和HO-1组海马CA1区ChAT表达降低，HO-1组海马CA1区HO-1表达升高（P〈0．05或0．01）；与IS组比较，HO-1组学习记忆能力提高，HO-1组海马CA1区ChAT和HO-1表达升高（P〈0．05）。结论 rAAV-rHO-1基因转染可抑制大鼠慢性脑缺血后认识功能减退，其机制与升高海马神经元ChAT的表达有关。

Objective To investigate the effect of transfection of heme oxygenase-1 （HO-1） gene mediated by recombinant adeno-associated virus vector （rAAV） （rAAV-HO-1） on cognitive function and the expression of choline acetyltransferase （CHAT） in hippocampus in rats with chronic cerebral ischemia. Methods Fifty-four male SD rats aged 10-11 months weighing 250-300 g were randomly divided into 3 groups （n = 18 each）： group Ⅰ sham operation （S）; gorup Ⅱchronic cerebral ischemia （CCI） and group Ⅲ HO-1 ＋ CCI （HO-Ⅰ）. The animals were anesthetized with intraperitoneal 10% chloral hydrate 3 ml/kg, rAAV-HO-1 gene 2μl （1 ×10^9 v.g. ） was injected into CA1 region of hippocampus in group Ⅲ （HO-1） while in group S （ Ⅰ ） and CCI （ Ⅱ ） 2 μl of normal saline was injected instead. Seven days after hippocampal injection bilateral common carotid arteries were permanently occluded by ligation in group Ⅱ and Ⅲ Two months later Morris water maze （MWM） test was performed to evaluate learning and memory ability. Fifteen animals were killed after MWM test in each group and their brains were removed for histologic examination of CA1 region of hippocampus with light and electron microscope and determination of the expression of ChAT and HO-1 in CA1 region of hippocampus. Results The ability of learning and memory was significantly lower in CCI group （ Ⅱ ） than in S group. The escape latency was significantly shorter in HO-1 group （ Ⅲ ） than in CCI group. The number of the neurons and synapses in hippocampus were significantly lower in CCI group than in S and HO-1 groups. The number of ChAT and HO-1 expression positive neurons in hippocampal CA1 region in CCI group was significantly decreased as compared with group S and was significantly larger in HO-1 group than in CCI group. Conclusion The transfection of HO-1 gene mediated by a recombinant adeno-associated virus vector can improve cognitive function in rats with chronic cerebral ischemia by increasing ChAT expression in hippocampal neurons.