目的 克隆芍药肌动蛋白（Actin）基因并应用RT-PCR技术分析Actin基因在芍药不同组织中的表达情况。方法 根据近缘物种Actin基因的保守序列设计一对PCR扩增引物，以“桃花飞雪”芍药根部总RNA反转录而成的cDNA为模板，采用RT-PCR的方法扩增出芍药Actin cDNA全长并克隆至pMD18-T载体上，阳性克隆经菌落PCR、质粒PCR及酶切鉴定后进行测序。基于测序结果设计特异性PCR引物，应用RT-PCR技术分析Actin基因在芍药根、茎、花、叶不同组织中的表达情况。结果 测序结果表明芍药Actin基因全长1 134 bp序列，共编码377个氨基酸，GenBank登录号为JX310002；序列分析表明芍药Actin蛋白中存在3种肌动蛋白特征信号序列，与其他植物肌动蛋白氨基酸同源性达99%；基于同源模建预测的3D结构中含有4个结构域；生物信息学软件预测芍药肌动蛋白的相对分子质量为4.17×10⁴，等电点（pI）为5.31，是一个定位于胞液、不含跨膜域、不含信号肽分子的亲水性、稳定蛋白；半定量RT-PCR技术分析表明Actin基因在芍药根、茎、叶、花组织中的表达量保持恒定。结论 首次从芍药中克隆了Actin基因，半定量RT-PCR表达分析结果表明Actin基因适合作为芍药功能基因表达分析的内标基因，为有效利用该基因奠定了基础。

Objective To investigate the effects of Tongsaimai Prescription (TSMP) and TSMP without Achyranthis Bidentatae Radix (ABR) on ischemic brain injuries in rats. Methods The male SD rats were divided into Sham, model, Nimodipine (32.4 mg/kg), TSMP (crude drug 31.74 g/kg), and TSMP without ABR (crude drug 27.77 g/kg) groups. Except the Sham and model groups, the rats were ig administered once daily, for 3 d. After the treatment, except the Sham group, the cerebral ischemic stroke model was established. After 24 h, the blood and brain tissue were taken, the brain tissue was stained with TTC, and the cerebral infarction ratio was measured. The pathological changes were observed, and the contents of HMGB1, TNF-α, and IL-1β in serum were detected. The expression of NF-κB in ischemia tissue was observed by immunohistochemisty method. Results The expression of HMGB1, TNF-α, and IL-1β increased obviously (P < 0.05). TSMP and TSMP without ABR could decrease the contents of HMGB1, TNF-α, and IL-1β, inhibit the expression of NF-κB (P < 0.05), and the effect of TSMP without ABR on decreasing IL-1β was stronger. Conclusion Both TSMP and TSMP without ABR could inhibit the activation of HMGB1-related NF-κB signal pathway with the function of anti-imflammation, alleviated encephaledema, and protecting nerve cells. TSMP without ABR has the similar effect on the ischemic brain injuries and the better effect on inhibiting the secretion of inflammatory factor IL-1β.

国家自然科学基金资助项目（81073071）；江苏省优势学科建设工程资助项目（2011ZYX3-0021）