补阳还五汤对脑缺血模型大鼠CyclinD1和CDK2表达及细胞增殖的影响

发布时间：2018-06-12 16:37

本文选题：脑缺血损伤 + 补阳还五汤　；参考：《湖南中医药大学》2013年硕士论文

【摘要】：目的：观察大鼠局灶性脑缺血后CyclinD1及CDK2的表达与细胞增殖的关系及补阳还五汤的干预作用,从细胞增殖角度探讨补阳还五汤的神经保护作用机制,为中医药治疗脑缺血性神经损伤提供新的认识,为其临床应用提供实验依据。方法：采用大脑中动脉线栓法(MCAO)建立局灶性脑缺血大鼠模型,将成年大鼠随机分为4组,即空白组,假手术组,模型组,补阳还五汤组,给予不同处理,分别于7天,14天,21天处死,对大鼠进行神经功能缺失症状评分,采用TTC法计算其梗死面积,免疫组化法观察缺血脑组织CyclinD1及CDK2的表达及BrdU标记的细胞新生情况。结果： 1.神经功能症状评分：正常组、术后2h假手术组大鼠未见神经功能缺损,造模后各手术组大鼠都存在有明显神经功能缺损；与假手术组比较,各时间点的模型组神经功能障碍评分显著增加(P0.01)；与模型组比较,用药7天,14天,21天后补阳还五汤组神经功能缺失症状评分减低(P0.05)；说明补阳还五汤对大鼠脑缺血损伤具有一定的保护作用。 2.梗死面积比的测定：正常组,假手术组均未出现梗死灶,与正常组及假手术组比,模型组大鼠TTC染色显示梗死灶非常明显(P0.01),梗死面积比随时间延长而减少。与模型组相比,用药7天,14天,21天后,补阳还五汤组可有效地减小缺血后梗死面积(P0.01),梗死面积随用药时间的增加而减少。 2. CyclinDl及CDK2的表达：正常组,假手术组可见少量的CyclinDl及CDK2蛋白的表达,脑缺血后皮层区与缺血半暗带区CyclinDl及CDK2阳性细胞大量表达(P0.01),7天达高峰,随时间延长有依次减少的趋势。用药7天,14天,21天后,补阳还五汤组能显著增加脑缺血后皮层区与缺血半暗带区CyclinDl及CDK2的表达,较模型组有显著差别(P0.01),随时间延长有依次减少的趋势。 3.BrdU阳性细胞测定：正常组,假手术组可见少量BrdU阳性细胞,模型组可见BrdU阳性细胞增加,有随时间增加的趋势,但是增长缓慢。用药7天,14天,21天后,补阳还五汤组能显著增加BrdU阳性细胞的数量,差异显著(P0.01),21天达高峰。相邻切片可见BrdU阳性细胞的表达区域与CyclinDl、CDK2的表达区域基本相同。结论： 1.大鼠局灶性脑缺血后脑组织因缺血造成严重的神经功能损伤和脑梗死,补阳还五汤可减轻缺血后的神经功能损伤,减少梗死面积。 2.大鼠局灶性脑缺血后皮层区与缺血半暗带区CyclinDl及CDK2的表达增加,补阳还五汤可增加皮层区与缺血半暗带区CyclinDl及CDK2的表达,早期效果明显。 3.大鼠局灶性脑缺血后BrdU有少量表达,补阳还五汤可显著增力口BrdU表达,通过调控CyclinDl、CDK2的表达使神经细胞增殖,从而诱导神经再生作用,起到神经保护作用。
[Abstract]:Objective: to observe the relationship between the expression of CyclinD1 and CDK2 and cell proliferation after focal cerebral ischemia in rats and to explore the neuroprotective mechanism of Buyang Huanwu decoction from the point of view of cell proliferation. To provide a new understanding for the treatment of cerebral ischemic nerve injury with traditional Chinese medicine, and to provide experimental basis for its clinical application. Methods: the rat model of focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO). Adult rats were randomly divided into four groups: blank group, sham operation group, model group and Buyang Huanwu decoction group. The rats were killed on the 7th day, 14th day and 21st day. The neurological deficit symptoms were scored, the infarct size was calculated by TTC method, the expression of CyclinD1 and CDK2 in ischemic brain tissue and BrdU labeled cells were observed by immunohistochemical method. Results: 1. The neurological function symptom score: normal group, 2 hours after operation group rats did not have nerve function defect, each operation group had obvious nerve function defect after modeling, compared with the sham operation group, Compared with the model group, the neurological dysfunction score of the model group increased significantly at each time point, and compared with the model group, the neurological deficit symptom score of the Buyang Huanwu decoction group decreased P0.05 after 7 days and 14 days and 21 days after treatment. It shows that Buyang Huanwu decoction has certain protective effect on cerebral ischemia injury in rats. 2. Compared with normal group and sham operation group, TTC staining of model group showed that the infarct area was very obvious (P 0.01), and the infarct area ratio decreased with time. Compared with the model group, the Buyang Huanwu decoction group could effectively reduce the infarct area after ischemia (P 0.01) and the infarct area decreased with the increase of medication time after 7 days and 14 days and 21 days later. Expression of Cyclin D1 and CDK2: a small amount of Cyclin D1 and CDK2 protein were found in normal and sham operation groups. The positive cells of cyclin D1 and CDK2 in cerebral ischemic cortex and ischemic penumbra reached the peak at 7 days after ischemia. After 7 days, 14 days and 21 days, Buyang Huanwu decoction group could significantly increase the expression of cyclin D1 and CDK2 in cerebral ischemic cortex and ischemic penumbra. Compared with the model group, there was a significant difference between the model group and the model group (P 0.01), and decreased with time. 3. BrdU positive cells were detected in the normal group and sham operation group, and BrdU positive cells were increased in the model group. But growth is slow. After 7 days, 14 days and 21 days, Buyang Huanwu decoction group could significantly increase the number of BrdU positive cells, and the difference reached the peak at 21 days. The expression of BrdU positive cells in adjacent sections was similar to that of Cyclin Dlt1 CDK2. Conclusion: 1. After focal cerebral ischemia, the brain tissue of rats suffered from severe nerve function injury and cerebral infarction due to ischemia. Buyang Huanwu decoction can reduce the nerve function injury and reduce the infarct area after ischemia. The expression of cyclin D1 and CDK2 in cortex and ischemic penumbra was increased after focal cerebral ischemia in rats. Buyang Huanwu decoction could increase the expression of cyclin D1 and CDK2 in cortex and ischemic penumbra. There was a little expression of BrdU after focal cerebral ischemia in rats, and Buyang Huanwu decoction could significantly increase the expression of BrdU in the mouth of rats. By regulating the expression of CyclinDltl CDK2, nerve cells proliferated, thus inducing nerve regeneration and playing a neuroprotective role.
【学位授予单位】：湖南中医药大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R285.5;R-332;R277.7

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