【作者】 李大伟；

【导师】 陈传民；

【作者基本信息】 泰山医学院 ， 内科学， 2012， 硕士

【摘要】 目的观察白花丹参干预对大鼠脑缺血损伤侧新生细胞增殖的影响，探讨脑缺血损伤及白花丹参干预对神经再生的影响及其可能机制，从而为白花丹参治疗缺血性脑血管病提供实验依据，为白花丹参的开发利用提供理论基础。方法1.局灶性脑缺血模型的建立：本实验采用经典的线栓法制备局灶性脑缺血再灌注模型（middle cerebral artery occlusion, MCAO）。所用实验动物为雄性成年SD（Sprague-Dawley）大鼠，实验麻醉剂选择10％的水合氯醛腹腔麻醉，麻醉后将特制的线栓经颈总动脉（common carotid artery, CCA）插入，沿颈内动脉（internal carotidartery，ICA）走行至至大脑中动脉起始部位及大脑前动脉近心端，从而阻断大脑中动脉的血流，使其所供应的脑区出现缺血，进线深度以CCA分叉开始计算大约为18.0±0.5mm。随着缺血时间的延长，其血流灌注部位的功能即出现障碍，如大鼠会出现偏瘫和偏身感觉障碍。手术完成后于脑缺血1h缓慢拔出栓线，同时结扎ECA残端以防止出血，实验大鼠于缺血再灌注后的不同时间点分别处死取脑。2．BrdU掺入方法：BrdU于动物处死取材前三天进行腹腔注射，注射剂量为100mg/kg，每天3次，最后一次注射2h后取材。3．白花丹参制剂制备：白花丹参根洗净凉干，用蒸馏水浸泡2h，煮沸30min，过滤，药渣加3～5倍量水，继续煎煮，煮沸20min后进行过滤，过滤后将滤液合并混合，滤渣加入适量乙醇混合后过夜沉淀并提取上清液，此过程需重复操作3遍，最后将所有滤液收集混合，混合后放于40℃真空中进行蒸发浓缩，浓缩后加入蒸馏水定容，放于4℃保存备用。4．免疫组织化学方法检测脑内BrdU阳性细胞雄性SD大鼠，随机分为三组：①假手术组；②单纯脑缺血再灌注组；③脑缺血再灌注＋白花丹参治疗组。脑缺血1h再灌注14d后取材，制备冰冻切片，采用免疫组织化学标记方法检测BrdU阳性细胞。5．水迷宫实验：雄性SD大鼠，随机分为三组：①假手术组；②单纯脑缺血再灌注组；③脑缺血再灌注＋白花丹参治疗组。对大鼠水迷宫的训练于术前5d即开始进行，所有实验大鼠于术前、脑缺血1h再灌注14d分别记录大鼠的逃避潜伏期（escapelatency, EL）。结果1．BrdU阳性细胞表达（1）脑缺血损伤侧纹状体区新生细胞表达：与假手术对照组比较，单纯脑缺血再灌注组新生细胞明显增多（P＜0.01），脑缺血再灌注＋白花丹参治疗组新生细胞较单纯脑缺血再灌注组新生细胞明显增多（P＜0.01）；（2）脑缺血损伤侧侧脑室区新生细胞表达：与假手术对照组比较，单纯脑缺血再灌注组新生细胞明显增多（P＜0.01），脑缺血再灌注＋白花丹参治疗组新生细胞较单纯脑缺血再灌注组新生细胞明显增多（P＜0.01）；（3）脑缺血损伤侧海马区新生细胞表达：与假手术对照组比较，单纯脑缺血再灌注组新生细胞明显增多（P＜0.01），脑缺血再灌注＋白花丹参治疗组新生细胞较单纯脑缺血再灌注组新生细胞明显增多（P＜0.01）；2.水迷宫实验：与假手术对照组比较，单纯脑缺血再灌注组EL明显延长（P＜0.01）；脑缺血再灌注＋白花丹参治疗组EL较单纯脑缺血再灌注组EL明显缩短（P＜0.01）。结论1．急性缺血性脑损伤可诱导大鼠损伤侧脑区新生细胞大量增殖。2．白花丹参干预可诱导脑内新生细胞增殖，新生增殖细胞在损伤侧大脑皮层、纹状体及海马表达较多。3．白花丹参干预后可使大鼠学习记忆能力明显加强。更多还原

【Abstract】 ObjectiveTo investigate the influence of Salvia miltiorrhiza Bunge f.alba on neurogenesis in ratsafter cerebral ischemia/reperfusion(I/R), and explore the possible mechanism and functionof Salvia miltiorrhiza Bunge f.alba in cerebral ischemic injury in order to provide theexperimental evidence for patients with ischemic cerebrovascular diseases.Methods1. Animal models preparation: The middle cerebral artery occlusion/reperfusion(MCAO) model of rats were established by the modified Longa occlusion method. Maleadult Sprague-Dawley (SD) rats were anesthetized with10﹪chloral hydrate byintraperitoneal injection. The right common carotid artery (CCA), internal carotid artery(ICA) and external carotid artery (ECA) were isolated via a cervical midline incision. Anylon suture was introduced into ECA and advanced into ICA,18.0±0.5mm in depth fromthe CCA branch, via middle cerebral artery, all the ways to the root of anterior cerebralartery (ACA), in order to block the blood of middle cerebral artery. After1hours ofischemia, the nylon suture was drawn out to restore reperfusion for14days. In shamoperation group, the nylon suture was inserted around ECA approaching the ICA branchwithout occlusion.2. BrdU injection: To begin to inject BrdU (100mg/kg) into the enterocoelia threedays before drawing materials, three times a day, with the last injection2hours earlier.3. Salvia miltiorrhiza Bunge f.alba solution preparation: The root was washed anddried, immersed for2hours, boiled for30minutes, then filtered. Three to five times waterwas added then, boiling for20minutes, filtered. The filtrate was then mixed with95%ethanol, precipitating one night for the filtrate. All the above was repeated for three times.The final filtrate was then condensed and reserved at4celsius degrees. 4. Immunohistochemistry to test BrdU positive cells in the brain: Male SD rats wererandomly divided into sham group, I/R group and I/R+Salvia miltiorrhiza Bunge f.albagroup. After ischemia1hour and reperfusion for14days, the rats were killed to preparefrozen sections. Then immunohistochemistry was used to detect BrdU positive cells.5. Water maze test: Five days before the operation, rats were trained for water maze,recording respectively the escape latency before and after ischemia1h/reperfusion14d.Result1. Expression of BrdU positive cells:（1）Neurogenesis in corpora striata of the injured side: The I/R group expressed morethan sham group (p<0.01), but less than I/R+Salvia miltiorrhiza Bunge f.alba group(p<0.01).（2）Neurogenesis in the lateral cerebral ventricle of the injured side: The I/R groupexpressed more than sham group (p<0.01), but less than I/R+Salvia miltiorrhiza Bungef.alba group (p<0.01).（3）Neurogenesis in hippocampus of the injured side: The I/R group expressed morethan sham group (p<0.01), but less than I/R+Salvia miltiorrhiza Bunge f.alba group(p<0.01).2. Water maze test: Escape latency(EL) of I/R group was longer than that of shamgroup (p<0.01), while that of Salvia miltiorrhiza Bunge f.alba was much shorter thanI/R(p<0.01).Conclusion1. Neurogenesis were increased after the acute focal cerebral ischemia injury in rats，brain.2. Based on the first conclusion, neurogenesis was considerably increased with the useof Salvia miltiorrhiza Bunge f.alba, especially in corpora striata, lateral cerebral ventricleand hippocampus.3. The use of Salvia miltiorrhiza Bunge f.alba strengthened the learning and memoryability of rats.更多还原