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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 aim of this study was to establish a method for the quantitative determination of IL-1β and Caspase-3 in rat cerebral ischemia / reperfusion (I / R) by real-time fluorescence PCR. Adult male Sprague-Dawely rats were randomly divided into 5 groups: sham operation group, I / R model group, Astragaloside group, ligustrazine-Astragaloside group, nimodipine group. Except for the sham operation group, the other groups were treated with brain I / R and then treated by intraperitoneal injection for 0h, 12h, 1d, 2d and 7d after brain I / R. The rats in the sham operation group and the I / R model group were injected with normal saline (5mL / kg), Astragaloside IV in the Astragaloside group was 20mg / kg, and the doses of ligustrazine and Astragaloside in the Ligustrazine-Astragaloside group were 10mg / kg and 20 mg / kg, while the nimodipine dose in the nimodipine group was 10 mg / kg. The IL-1β and Caspase-3 genes were cloned by RT-PCR. The recombinant plasmids pTA2-IL1 and pTA2-Casp3 were constructed by TA cloning technique. A260 / A280 ratio was determined by ultraviolet spectrophotometer Calculate the copy number. Using the plasmid as a standard, the primers of the real-time PCR reaction were screened and validated first, and then the temperature of the reaction annealing was optimized. Finally, the gene expression of IL-1β and Caspase-3 in brain tissues were detected quantitatively. The results showed that a single peak was obtained from the melting curve analysis of each pair of the best primer selected from the candidate primers. The agarose gel electrophoresis showed that the reaction product was consistent with the expected target product size. Gradient annealing temperature experiments showed that the optimal annealing temperature of IL-1β and Caspase-3 genes were 59 ° C and 61.2 ° C, respectively. The results of real-time fluorescence PCR showed that the expression of IL-1β and Caspase-3 in I / R model group was significantly higher than that in sham operation group. Compared with I / R model group, Astragaloside group and nimodipine group IL-1β and Caspase-3 gene expression levels were decreased, especially tetramethylpyrazine - Astragaloside group gene down-regulated the most significant. The above results suggest that the method established in this study is suitable for the quantitative analysis of IL-1β and Caspase-3 gene after the Chinese medicine treatment of I / R model.