丁苯酞动员内皮祖细胞治疗缺血性卒中大鼠的实验研究

发布时间：2018-04-27 00:38

  本文选题：缺血性卒中 + 内皮祖细胞　； 参考：《苏州大学》2014年硕士论文

【摘要】：目的：观察不同剂量丁苯酞对大脑中动脉缺血（MCAO）/再灌注模型大鼠神经功能、循环内皮祖细胞(circulating endothelial progenitor cells, EPCs)水平及梗死区血管新生的影响，探讨其治疗缺血性卒中的疗效及机制。 方法：选取健康雄性大鼠Sprague-Dawley（SD）大鼠48只，体重（250±20）g， 按照随机分组法分为假手术组，模型对照组，NBP低剂量治疗组（60mg/kg.d）和NBP高剂量治疗组(120mg/kg.d），每组各12只。假手术组仅分离颈部血管而不插入线栓。其他三组均采用线栓法建立大鼠MCAO/再灌注模型，假手术组与模型组各取2只行TTC染色法观察大鼠梗塞区形态学改变，其余大鼠分别给予安慰剂(大豆油)（2ml/d）及两种剂量的NBP灌胃治疗，，持续5d。每天对四组大鼠进行神经功能缺损评分；给药前及灌胃5d后分两次采集大鼠外周血，流式细胞仪检测治疗前后循环EPCs的数量；灌胃5d后行血管内皮细胞标记物CD31及vWF免疫组化染色，观察各组大鼠缺血区血管新生情况。GraphPad5.0软件系统对实验数据进行统计分析处理。 结果： 1）脑TTC染色后可见线栓侧大脑半球明显梗死病灶，呈苍白色，非梗死区显示为红色； 2）NBP治疗第1d，低剂量组大鼠神经功能评分开始出现降低（P0.05），高剂量组神经功能评分明显降低（P0.01），模型对照组大鼠神经功能评分在灌胃3d后出现明显降低（P0.01）； 3）NBP治疗第1d，低剂量组大鼠神经功能评分低于对照组（P0.05），高剂量组神经功能评分明显低于对照组（P0.01）；第2-5d，NBP两种剂量治疗组大鼠神经功能评分均显著低于对照组（P0.01）； 4）NBP治疗第3d，高剂量组神经功能评分明显低于低剂量组（P0.01）； 5）灌胃5d后与缺血2h再灌注时比较，假手术组大鼠外周血EPCs数量无明显变化，其余各组均显著增加（P0.01）； 6）不同剂量NBP治疗组大鼠在5d后的外周血EPCs数量均显著高于对照组（P＜0.01）；NBP高剂量组外周血EPCs水平显著高于低剂量组（P0.01）； 7）假手术组脑切片显微镜下可见抗CD31阳性细胞和抗vWF阳性细胞，呈正常脑组织内血管染色；对照组脑切片几乎未见或极少量表达抗CD31阳性或抗vWF阳性细胞，提示脑缺血区血管破坏；NBP治疗组脑切片在光镜下均可看到抗CD31阳性及抗vWF阳性细胞大量表达，其中NBP高剂量治疗组的阳性细胞表达更为显著。 结论： 1）MCAO/再灌注损伤可促进EPCs动员； 2）丁苯酞可进一步增强循环EPCs动员，有效地促进缺血区血管新生，改善神经功能缺损评分；高剂量较低剂量疗效更显著。
[Abstract]:Objective: to observe the effects of different doses of butyphthalide on nerve function, circulating endothelial progenitor cell (endothelial progenitor cells, EPCs) level and angiogenesis in infarcted area of rats with middle cerebral artery ischemia (MCAO) / reperfusion, and to explore the therapeutic effect and mechanism of butyphthalide on ischemic stroke. Methods: a total of 48 healthy male Sprague-Dawley SD rats were selected with a body weight of 250 卤20g. According to the method of random grouping, the model group was divided into sham operation group, model control group, low dose treatment group (60 mg / kg 路d) and NBP high dose group (120 mg / kg 路dg / d), with 12 rats in each group. In the sham operation group, only cervical vessels were separated and no thread embolus was inserted. The other three groups were used to establish MCAO/ reperfusion model in rats. Two rats in sham-operation group and two in model group were taken to observe the morphological changes of infarct area by TTC staining. The rest of the rats were given placebo (soybean oil 2 ml / d) and two doses of NBP for 5 days. The peripheral blood was collected twice before and after 5 days of administration, and the number of circulating EPCs before and after treatment was detected by flow cytometry. After 5 days of gastric perfusion, the vascular endothelial cell markers (CD31 and vWF) were stained by immunohistochemistry. The angiogenesis in ischemic area of rats in each group was observed. The experimental data were statistically analyzed by GraphPad 5.0 software system. Results: 1) after TTC staining, the cerebral hemispheres showed obvious infarct lesions, which were pale and red in non-infarcted areas. On the 1st day after 2)NBP treatment, the neurological function scores of the rats in the low dose group began to decrease P0.05A, in the high dose group decreased P0.01a significantly, and in the model control group, the neurological function scores of the rats in the model control group decreased significantly after 3 days of gastric administration. On the 1st day of 3)NBP treatment, the scores of nerve function in the low dose group were lower than those in the control group (P 0.05), the scores in the high dose group were significantly lower than those in the control group (P 0.01), and the scores of nerve function in the 2 to 5 days treatment group were significantly lower than those in the control group (P 0.01). On the 3rd day after 4)NBP treatment, the scores of nerve function in the high dose group were significantly lower than those in the low dose group (P 0.01). 5) after 5 days of gastric perfusion, there was no significant change in the number of EPCs in peripheral blood of rats in sham-operated group compared with that at 2 h after ischemia and reperfusion. 6) the number of EPCs in peripheral blood of rats treated with different doses of NBP after 5 days was significantly higher than that of control group (P < 0.01). The level of EPCs in high dose group was significantly higher than that in low-dose group (P 0.01). 7) Anti CD31 positive cells and anti vWF positive cells were observed under microscope in sham-operation group, and showed vascular staining in normal brain tissue, while no or very few anti CD31 positive or anti vWF positive cells were found in brain sections of the control group. It was suggested that the positive cells of anti- and anti- could be seen in the brain sections of the cerebral ischemic area treated with NBP under light microscope, especially in the high dose group of NBP. The results showed that the expression of anti-NBP positive cells was more significant in the high-dose NBP group than in the high-dose NBP group. Conclusion: 1)MCAO/ reperfusion injury can promote EPCs mobilization. 2) Butylphthalide could further enhance the mobilization of circulating EPCs, promote angiogenesis in ischemic area and improve the score of nerve function defect, and the effect of high dose was more obvious than that of low dose.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】

相关期刊论文 前7条

1 曹文英;德吉曲宗;李其富;何俐;周东;;丁基苯酞对大鼠短暂性缺血脑组织VEGF及bFGF表达的影响[J];四川大学学报(医学版);2009年03期

2 焦东亮;倪秀石;高艳;赵杨;夏煜;;丁苯酞对大鼠局灶性脑缺血再灌注损伤后caspase-3表达的影响[J];中国临床神经科学;2007年01期

3 崔丽英,李舜伟,吕传真,董强,董鹏,史朗峰,黄鉴政,毛善英,张朝东,曹云鹏,聂莹雪,王维治,梁庆成,杨春晓,董为伟,李光勤,彭国光,罗祖明,何俐,万琪,韩军良,黄如训,李玲,李承宴,蒙振中;恩必普软胶囊治疗中度急性缺血性卒中的多中心开放临床研究[J];中国脑血管病杂志;2005年03期

4 ;Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury[J];Neural Regeneration Research;2012年12期

5 种兆忠,冯亦璞;丁基苯酞减轻大鼠局灶性脑缺血再灌注引起的血脑屏障损伤<英文>[J];Acta Pharmacologica Sinica;1999年08期

6 崔丽英,刘秀琴,朱以诚,樊东升,谢汝萍,沈杨,张微微,杨新平,哈志远,李玲,冯亚青,李舜伟;dl-3-正丁基苯酞治疗中度急性缺血性脑卒中的多中心、随机、双盲和安慰剂对照研究[J];中华神经科杂志;2005年04期

7 董高翔,冯亦璞;丁基苯酞对局部脑缺血再灌注大鼠脑线粒体ATPase,抗氧化酶活性和脂质过氧化的影响[J];中国医学科学院学报;2002年01期

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