川芎嗪-黄芪甲苷降低缺血/再灌注损伤的大鼠脑组织中IL-1β和Caspase-3基因的表达：实时定量PCR的研究(英文)

Chuanxiongzine-astragaloside IV decreases IL-1β and Caspase-3 gene expressions in rat brain damaged by cerebral ischemia/reperfusion:A study of real-time quantitative PCR assay

本文旨在运用实时荧光PCR技术建立大鼠脑缺血/再灌注(ischemia-reperfusion,I/R)中IL-1β和Caspase-3基因绝对定量分析方法。成年雄性Sprague-Dawely大鼠被随机分成了5组:假手术组、I/R模型组、黄芪甲苷组、川芎嗪-黄芪甲苷组、尼莫地平组。除假手术组外,其余各组均进行脑I/R处理,然后通过腹膜内注射进行药物处理,时间为脑I/R后0h、12h、1d、2d直至7d。假手术组和I/R模型组注射生理盐水(5mL/kg),黄芪甲苷组中黄芪甲苷剂量为20mg/kg,川芎嗪-黄芪甲苷组中川芎嗪和黄芪甲苷剂量分别为10mg/kg和20mg/kg,而尼莫地平组中尼莫地平剂量为10mg/kg。通过常规RT-PCR克隆了大鼠的IL-1β和Caspase-3基因,运用TA克隆技术分别构建了重组质粒pTA2-IL1和pTA2-Casp3,重组质粒经过紫外分光光度计测定A260/A280比值,并计算拷贝数。以该质粒作为标准品,首先对实时荧光PCR反应的引物进行筛选和验证,然后进行反应退火温度的优化,最后定量检测各组损伤的脑组织中IL-1β和Caspase-3的基因表达情况。结果显示,从候选引物中各筛选到一对最佳引物,熔解度曲线分析显示单一峰,琼脂糖电泳表明反应产物与预计目标产物大小一致。梯度退火温度实验表明IL-1β和Caspase-3基因的最佳退火温度分别是59°C和61.2°C。实时荧光PCR检测结果表明,与假手术组相比,I/R模型组中的IL-1β和Caspase-3表达显著升高;和I/R模型组相比,黄芪甲苷组、川芎嗪-黄芪甲苷组、尼莫地平组中IL-1β和Caspase-3基因表达水平均有所下降,特别是川芎嗪-黄芪甲苷组基因下调最显著。以上这些结果提示,本研究建立的方法适用于中药处理I/R模型后IL-1β和Caspase-3基因的定量分析。

The purpose of this study was to establish an absolute quantitative method to detect IL-1β and Caspase-3 gene expressions in rat brain after cerebral ischemia-reperfusion（I/R） using real-time PCR.Rats were randomized into the following groups：sham operation group,model group（cerebral I/R group）,astragaloside IV（AST IV） group,chuanxiongzine-AST IV group and nimodipine group（n = 10 in each group）.The rats in all the groups except sham operation group were subjected to cerebral I/R treatment.Sham operation and model groups were treated by normal saline（5 mL/kg）.AST IV,chuanxiongzine-AST IV and nimodipine groups received 20 mg/kg AST IV,10 mg/kg chuanxiongzine plus 20 mg/kg AST IV,and 10 mg/kg nimodipine treatments,respectively.The administrations of drugs were performed with intraperitoneal injections at 0 and 12 h,1 d,2 d,3 d,till to 7 d after I/R.A real-time quantitative PCR assay was developed for absolute quantification of the expressions of IL-1β and Caspase-3 genes.The absolute quantification approach relies on the construction of an accurate standard curve.Thus,two plasmids which contained rat IL-1β and Caspase-3 genes respectively were constructed.The cloned circular plasmids were then quantified using a spectrophotometer and used as standards.Standard curves were generated,and the copy numbers of IL-1β and Caspase-3 mRNA isolated from I/R-damaged brain tissue were also calculated by SYBR Green I dye method using specific primers.The results showed that melting curves exhibited sharp peaks,and PCR product also generated prominent band with expected size in agarose gel electrophoresis,which validated the optimization of the selected primer sets of IL-1β and Caspase-3 genes.The optimal annealing temperatures of IL-1β and Caspase-3 genes were 59 °C and 61.2 °C,respectively.Real-time PCR results showed that the expression of IL-1β and Caspase-3 genes in the model group was significantly elevated compared to that in the sham operation group.However,compared to those in the model group,IL-1β and Caspase-3 gene expressions were obviously decreased in AST IV,chuanxiongzine-AST IV and nimodipine groups.Especially in chuanxiongzine-AST IV group,those two genes showed the most significant expression down-regulation.These results suggest the absolute quantitative method established in the present study is capable of detecting the changes of IL-1β and Caspase-3 gene expressions in rat brain damaged by I/R.