局灶性脑缺血再灌注后Fos、Jun蛋白与Bcl-2、Bax蛋白的表达及益气、活血中药对其表达的影响

【作者】 曲友直；

【导师】 高国栋；

【作者基本信息】 第四军医大学 ， 外科学（神外）， 2001， 硕士

【摘要】 目的： 程序性细胞死亡（programned cell death，PCD）是脑缺血再灌注损伤过程中神经元死亡的一种形式，特别在迟发性神经元死亡过程中起十分重要的作用。目前研究认为PCD与即早基因（immediate-early genes，IEGS）及凋亡相关基因bcl-2家族表达的变化密切相关。中医学认为，缺血性卒中的病机主要是气虚血瘀，临床多采用益气活血方作为主要治则，疗效显著，但其作用机理尚未完全阐明。本研究通过观察益气活血药对即早基因蛋白Fos、Jun及凋亡相关蛋白Bcl-2、Bax表达的影响，探讨益气活血药对缺血性脑卒中起保护作用的机制。 方法： 采用颈外动脉栓线法制备局灶性脑缺血再灌注模型。①制备局灶性脑缺血90min、再灌注60min模型。缺血前30min经大鼠腹腔注射丹参、黄芪及丹参与黄芪注射液。采用免疫组化与医学图像分析相结合方法观察缺血侧大脑皮层Fos蛋白、Jun蛋白阳性细胞的数目及平均灰度。②制备局灶性脑缺血2h、再灌注6h模型。缺血前30min经腹腔注射川芎嗪、黄芪及川芎嗪与黄芪注射液。采用免疫组化与医学图像分析相结合的方法观察大鼠缺血侧大脑皮层Bcl-2蛋白、Bax蛋白阳性细胞数目及平均灰度变化。 簧四军医大学砰士学岱分文 结果： 1．模型组较假手术组Fos蛋白、Jun蛋白表达显著增强；丹 参、黄蔑及两药合用均可部分下调脑缺血再灌注诱导的F加蛋 白、Jun蛋白高表达，其中两药合用比单用丹参或黄蔑下调Fos 蛋白、Jun蛋白表达更显著。 2．模型组较假手术组B＊ 蛋白、B：X蛋白表达显著增强： 川穹嚎、黄蔑及两药合用均可上调脑缺血再灌注诱导的B＊E蛋 白的表达，下调Bax蛋白的表达，其中两药合用作用最显著。 结论： 1．局灶性脑缺血再灌注后Fos蛋白、Jun蛋白及BclZ蛋白、 Bax蛋白表达显著增强，表明脑缺血再灌注可诱导即早基因蛋白 及凋亡相关蛋白表达，即早基因蛋白及凋亡相关蛋白在脑缺血再 灌注后PCD中起重要作用。 2．本研究表明活血药丹参、益气药黄蔑及两药合用均可部分 下调Fos蛋自、Jun蛋白的高表达，对脑缺血再灌注损伤起保护 作用。活血药）11穹嗓、益气药黄蔑及两药合用均可上调脑缺血再 灌注诱导的BclZ蛋白表达，同时下调Bax蛋白表达，通过抗凋 亡发挥其脑保护作用。 3．本研究发现活血药与益气药合用对基因蛋白的影响优于 单用活血药或单用益气药，证明两类药物有明显协同作用。活血 化瘀配伍益气升阳类药物对即早基因蛋白及凋亡相关蛋白表达 的影响可能是其临床治疗缺血性卒中的分子机制之一。更多还原

【Abstract】 Purpose:Programmed cell death(PCD)was one of ways that neurons died after cerebral ischemia and reperfusion. PCD took very important effect on neurons’ death in delayed neuronal death (DND). It had been reported that expression of immediate-early genes(IEGS) and apoptosis-related genes were closely associated with PCD. In theories of Traditional Chinese Medicine (TCM), the mechanism of ischemic stroke was deficiency of Qi and blood stasis. Prescription of reinforcing Qi and activating blood had been using in clinic for a long time. It had positive effects significantly, but the mechanism hadn’t been known. The mechanism of drugs of reinforcing Qi and activating blood to protect neurons after ischemic stroke was investigated by means of effects of drugs of reinforcing Qi and activating blood on the expression of Fos protein > Jun protein > Bcl-2 protein and Bax protein.Methods:String inserting method through external carotid artery was used to make the model of focal cerebral ischemia and reperfusion. ㏑adix salviea miltiorrhizae(RSM)> astragus and prescript of RSM and astragus were injected through abdominal cavity before ischemia in rats. The immunohistochemistry and MIPS were used to measure number and mean grey level of positive cells of Fosprotein , Jun protein.2 Ligustrazine (LZ), astragus and prescript of LZ and astragus were injected through abdominal cavity before ischemia. The immunohistochemistry and MIPS were used to measure number and mean grey level of positive cells of Bcl-2 protein, Bax protein in rats’ cortex of ischemic sides. Results:1. The expression of Fos protein and Jun protein in model group increased significantly, compared with sham-operated group; RSM, astragus , RSM and astragus all could inhibit partly the expression of Fos protein , Jun protein after cerebral ischemia and reperfusion; Prescription of RSM and astragus had stronger inhibiting effects than RSM or astragus.2. The expression of Bcl-2 proteinN Bax protein in model group increased signicantly, compared with sham-operated group; LZ, astragus , LZ and astragus all could increase the expression of Bcl-2 protein and inhibit the expression of Bax protein at the same time; prescription of LZ and astragus had stronger effects than LZ or astragus.Conclusion:1. The expression of Fos protein , Jun protein Bcl-2 protein and Bax protein increases significantly after focal cerebral ischemia and reperfusion. It suggests that the exprssion of IEGS and apoptosis-related genes can be induced by cerebral ischmia and reperfusion. It is verified that IEGS and apoptosis-related genesplay important roles in PCD.2. The drug of activating blood , the drug of reinforcing Qi and the prescript of activating blood and reinforcing Qi all could partly inhibit expression of Fos protein or Jun protein to protect neurons after cerebral ischemia and reperfusion. The drug of activating blood, the drug of reinforcing Qi and the prescript of activating blood and reinforcing Qi all could increase the expression of Bcl-2 protein and decrease the expression of Bax protein to inhibit apoptosis and atteunate brain injury after cerebral ischemia and reperfusion.3. The prescript of reinforcing Qi and activating blood has stronger effects on the expression of IEGS proteins and apoptosis related proteins than either drug of reinforcing Qi or drug of activating blood. It is verified that there is synergism action between the drug of activating blood and the drug of reinforcing Qi. It may be one of mechanisms of reinforcing Qi and activating blood circulation therapy to treat ischemic stroke in clinic.更多还原