早期给予黄芪注射液治疗大鼠脑出血对血肿周围胞凋亡的影响(1)

    【关键词】 脑出血；黄芪注射液；细胞凋亡；大鼠

     The effect of astragalus propinquns injection on cell apoptosis around the hematoma in the early time after the experimental intracerebral hemorrhage in rats

    【Abstract】 Objective To study the effect of astragalus propinquns injection on apoptotic cell and caspase-3 protein in the early time after the experimental intracerebral hemorrhage（ICH） in rats. Methods All the ICH rats about the experiment were randomly divided into the remedial group injecting astragalus propinquns and the group without injection. Then the remedial group of rats were divided into four groups which were the instantly treated group， the 6 hours treated group， the 12 hours treated group and the 24 hours treated group. The remedial groups were injected with astragalus propinquns injection once a day. After that all ICH rats were killed on different days to take out their brain in which the immunohistochemical method was performed by using the antibody of caspase-3 protein and TUNEL method was used to detect apoptosis around the hematoma in the brain. To observe the effect of astragalus propinquns injection in different time on the neurological dysfunction， caspse-3 protein expression and cell apoptosis around the hematoma of the brain.Results Compared with the hemorrhage group， the apoptotic rate and the positive cells of caspase-3 protein in the remedial groups were significantly decreased (P<0.01) and neurological dysfunctions were significantly improved .The apoptotic rate of the instantly treated group and 6 hours later treated group decreased significantly compared with that of the 24 hours later treated group(P<0.01).Conclusion When astragalus propinquns injection was used earlier to cure ICH， it can decrease the rate of cell apoptosis and caspase-3 protein expression around the hematoma significantly.

    【Key words】 brain hemorrhage; astragalus propinquns injection;cell apoptosis;

    脑出血是一种严重危害人类生命健康的临床常见病和多发病。据统计，在我国，脑出血占急性脑血管病（脑卒中）的40%～50%，是脑卒中死亡率最高的临床类型，脑出血后30天内病死率接近50%，许多存活者遗留明显的神经功能缺失［1］。目前对高血压脑出血的治疗方法虽较以往有很大进步，如超早期手术、微创技术清除血肿和综合治疗等［2］，但由于影响脑出血疗效的因素是多方面的，脑出血仍具有相当高的死亡率。近年来， 有人尝试采用活血化瘀药黄芪治疗急性期的脑出血，取得了一定的效果［3］，但目前对于该药治疗脑出血的时机和机制问题尚不清楚。基于此问题，本实验欲在不同时间给予黄芪注射液，采用TUNEL染色和Caspase-3免疫组化染色的方法，观察大鼠神经功能缺损、血肿周围细胞凋亡及凋亡相关蛋白Caspase-3指标，以期达到早期应用黄芪治疗脑出血后观察其疗效的目的。

    1 材料与方法

    1.1 材料

    1.1.1 动物 SD大鼠雌雄不限200只，体重300～350g，由西安交通大学医学院实验动物中心提供，动物合格证号（医动字 第08-005号）。

    1.1.2 主要试剂 黄芪注射液(哈尔滨圣泰制药有限公司，批号Z23020820)；Ⅶ型胶原酶(sigma公司)；TUNEL荧光检测试剂盒（Promega公司），Caspase-3多克隆抗体（北京中衫生物公司），DAB染色试剂盒（武汉博士德公司）。

    1.2 方法

    1.2.1 动物分组 将大鼠（雌雄不限）随机分成4个治疗组：手术同时治疗组、术后6h治疗组、术后12h治疗组、术后24h治疗组和1个出血对照组。各治疗组每日给予腹腔注射黄芪注射液1次，剂量为8ml/（kg·d）（1ml=2g原药材），出血对照组不予治疗。每日进行神经功能评分1次，治疗组分别于第4天和7天处死。

    1.2.2 脑出血大鼠模型制作 根据脑立体定位图谱于大鼠前囟后0.2 ㎜，中线右侧旁开3.0㎜钻孔，暴露硬脑膜。微量进样器吸取1.0μg /μlⅦ型胶原酶0.25μl，以硬脑膜平面为零点，缓慢进针，深度为5.0 ㎜，缓慢注入药物，注射完毕后留针10 min，缓慢退出微量进样器，清洁伤口，缝合头皮，术后保温。

    1.2.3 免疫组织化学染色 石蜡切片，采用ABC法，DAB显色，光学显微镜下观察。在200倍视野下，随机观察并计数血肿周边3个不重复视野的Caspase-3蛋白免疫反应阳性细胞数目。

    1.2.4 细胞凋亡的检测 取与免疫组化染色相邻切片，用末端脱氧核糖核苷转移酶介导的dUTP缺口末端标记法(TUNEL)。在暗室内荧光显微镜下观片，在200倍视野下，随机观察并计数血肿周边3个不重复视野的TUNEL反应阳性细胞数目。

    1.2.5 结果分析 结果用x±s表示。统计学处理采用SPSS11.5软件包进行方差分析和相关分析，显著性水准均为α=0.05。

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