苦碟子注射液作用后缺血性脑卒中火毒证大鼠脑组织双向电泳技术的建立与优化

Establishment and optimization of two-dimensional gel electrophoresis for brain tissue in rats with heat-evil syndrome of cerebral ischemic stroke after Kudiez injection

目的建立和优化苦碟子注射液作用后缺血性脑卒中火毒证大鼠脑组织蛋白质组双向电泳技术。方法采用标准裂解法提取苦碟子注射液作用后缺血性脑卒中火毒证大鼠病灶侧梗死周边区大脑皮层组织总蛋白,分别利用三氯乙酸-丙酮沉淀法和去脂蛋白纯化法处理蛋白质样品,进行双向凝胶电泳(two-dimensional gel electrophoresis,2-DE),并对蛋白上样量和等电聚焦参数进行优化,考马斯亮蓝染色后对凝胶图像进行比较。结果去脂蛋白纯化法所得蛋白的2-DE图谱蛋白点数目明显增加,蛋白纯化效果较好,具有更好的溶解性和重复性;等点聚焦参数经改进后,所得图谱的分辨率较高,分离更完全;样品上样量为1 000μg时较适合,图谱蛋白点数目多、形状圆滑,且仅有几个高丰度蛋白出现点饱和。结论成功建立了苦碟子注射液作用后缺血性脑卒中火毒证大鼠脑组织蛋白质提取纯化方法,优化了双向电泳体系,从而获得更清晰的2-DE图谱,为后续研究奠定了技术基础。

Objective To establish and optimize two-dimensional gel electrophoresis for proteome of brain tissue in rats with fire-evil syndrome of cerebral ischemic stroke after Kudiez injection. Methods Standard pyrolysis method was used to acquire the total protein of cerebral cortex around infarction lesions in rats with heat-evil syndrome of cerebral ischemic stroke after Kudiez injection. The TCA /Acetone precipitation method and protein delipidation-purification method were respectively utilized to precipitate the protein sample to perform two-dimensional gel electrophoresis（ 2-DE）; and the loading quantity of protein sample and isoelectric focusing（ IEF） were adjusted and optimized. Modified Coomassie brilliant blue（ m CBB） was used to compare images of gels. Results In the 2-DE images of protein acquired by delipidation-purification method,the number of protein spot was increased significantly,and effect of protein purification was satisfied,indicating a better resolution and reproducibility. After improvement on IEF,the image had higher resolution and complete separation. The loading quantity of sample was appropriate with 1 000 μg,which would show more protein spots that are smooth and few high-abundant protein spots showing saturation. Conclusion The extraction and purification method is successfully established for brain tissue in rats with heat-evil syndrome of cerebral ischemic stroke after Kudiez injection,and two-dimensional gel electrophoresis system is optimized to acquire better 2-DE images,which could provide technique foundation for further study.