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目的探讨慢性低灌注大鼠海马CA1区胆碱乙酰转移酶(ChAT)的蛋白表达以及基因转录水平的改变,并观察异亚丙基莽草酸(ISA)对其影响。方法实验动物分为假手术组、模型组、喜得镇组、ISA100 mg/kg组、ISA50 mg/kg组以及ISA25 mg/kg组。将大鼠双侧颈总动脉结扎加切断后8周,经心灌注固定,制成大脑石蜡切片后进行免疫组织化学染色,测定海马CA1区ChAT定位以及相对定量表达。抽提大鼠海马CA1区组织总RNA,通过荧光定量实时PCR技术,对ChATmRNA表达水平进行测定。结果慢性低灌注65 d后,大鼠海马CA1区ChAT免疫反应阳性产物主要分布于锥体细胞的胞浆及轴突。模型组大鼠的ChAT蛋白表达水平以及ChATmRNA表达水平非常显著低于假手术组(P<0.05)。与模型组相比,各给药组的ChAT蛋白表达水平不同程度升高(P<0.05);ISA100 mg/kg组的ChATmRNA表达水平增加非常显著(P<0.05),而ISA其余各剂量以及喜得镇对ChATmRNA表达水平无显著影响(P>0.05)。结论ISA可增加慢性低灌注大鼠海马CA1区ChAT的蛋白以及基因表达水平。
Objective To investigate the protein expression of choline acetyltransferase (ChAT) in hippocampal CA1 region of chronic hypoperfusion rats and the changes of gene transcription, and to observe the effect of isopropylidene oxalate (ISA) on it. Methods Experimental animals were divided into sham operation group, model group, Xideng group, ISA 100 mg/kg group, ISA 50 mg/kg group and ISA 25 mg/kg group. Rats were subjected to bilateral ligation of the common carotid artery and ligated for 8 weeks. After perfusion and fixation, cerebral paraffin sections were prepared and immunohistochemically stained to determine ChAT localization and relative expression in the hippocampal CA1 region. Total RNA from rat hippocampal CA1 region was extracted and the expression level of ChAT mRNA was determined by real-time PCR. Results After chronic hypoperfusion for 65 days, the positive ChAT immunoreactive products in hippocampal CA1 region mainly distributed in cytoplasm and axons of pyramidal cells. The expression of ChAT protein and the expression of ChAT mRNA in the model group were significantly lower than those in the sham group (P<0.05). Compared with the model group, the expression level of ChAT protein was increased in different degrees (P<0.05); the expression level of ChAT mRNA in ISA 100 mg/kg group was significantly increased (P<0.05), while the remaining doses of ISA and hi Dezhen had no significant effect on the expression of ChAT mRNA (P>0.05). Conclusion ISA can increase the expression of ChAT protein and gene expression in hippocampal CA1 region in chronic hypoperfusion rats.