摘要
目的:建立一个方便、有效的动物实验模型以研究神经干细胞移植治疗脑缺血的可行性。方法:实验于2004-09/2005-10在第四军医大学全军神经外科研究所完成。健康家猫12只,随机分为缺血再灌注组(10只)和假手术组(2只),参照四血管阻塞法制作猫全脑缺血模型。先用显微手术的方法分离家猫双侧椎动脉并电凝,永久性闭塞双侧椎动脉。24h后再分离双侧颈总动脉,用动脉夹阻断血流。用含表皮生长因子的无血清培养基原代分离培养小鼠神经干细胞。将原代培养的小鼠神经干细胞移植到全脑缺血再灌注损伤模型的一侧大脑皮层中,假手术组猫大脑皮层注射同量的磷酸盐缓冲溶液;用免疫荧光技术检测移植1个月后的小鼠神经干细胞。结果:实验家猫12只,进入结果分析9只。①缺血再灌注组10只家猫经缺血再灌注损伤后死亡2只,1只夹闭血管后始终未出现静息脑电波,模型制作成功7只,成功率70%。假手术组家猫全部存活。缺血再灌注损伤后的家猫均出现不同程度的肢体瘫痪及平衡失调表现。饲养1周后,肢体瘫痪及平衡失调表现均逐渐有所改善。②原代培养细胞在无血清培养基中生长2～3d后,大部分细胞死亡,只有少数以单细胞形式悬浮生长。部分细胞分裂形成几个到几十个细胞的细胞团。到7～10d时可生长成有数十个到数百个细胞的细胞团,也呈悬浮生长。这些悬浮生长的细胞在含血清培养基中能够贴壁生长并部份分化成神经微丝抗原阳性的神经元及胶质纤维酸性蛋白抗原阳性的神经胶质细胞。③细胞移植后,家猫症状有所改善,但移植侧与对侧肢体瘫痪及平衡失调表现改善无明显差别。④免疫荧光方法检测小鼠神经干细胞移植到猫前脑后1个月仍有大量存活,部份细胞分化为神经元及神经胶质细胞,部份细胞向猫脑组织中迁移。迁移深度0.1～0.2mm。呈弥散性分布。沿针道侧壁贴附的细胞呈圆形,迁移到脑组织深部的细胞大多呈梭形。海马及对侧脑皮层等远隔部位未检测到阳性细胞存在。不同个体间未发现明显差异。⑤缺血再灌注组脑皮层及海马CA1区苏木精-伊红染色示典型的迟发性神经元坏死。结论:小鼠神经干细胞在猫全脑缺血模型中能够存活、迁移并分化;成功建立一个神经干细胞移植治疗脑缺血的实验模型。
AIM： To establish a convenient and effective animal experimental model in order to study the therapic feasibility of brain ischemia with neural stem cells graft. METHODS： This experiment was conducted at the Institute of Neurosurgery, Fourth Military Medical.University of Chinese PLA from September 2004 to October 2005. Twelve healthy cats were randomly divided into ischemic reperfusion group （n=10） and sham-operation group （n=2）.Accord- ing to four vessels occlusion methods, global ischemia models were established in ten cats.The method of microsurgical operation technique was used to separate bilateral vertebral artery of the cats.＂ Bilateral vertebral artery was occluded perpetually following electric coagulation. 24 hours later, bilateral common carotid artery was separated and the blood flow ＂was blocked by bulldog clamp. Neural stem cells of mouse were cultured primarily in medium with serum-free culture medium containing epidermal growth factor. Neural stem cells of the mouse＇cultured primarily was transplanted into cerebral cortex of one side of global brain ischemia models, and the same dose of phosphate buffer solution was injected into the cerebral cortex of cats in the sham-operation group; One month after transplantation, neural stem cells of the mouse was detected with immunofluorescenee technique. RESULTS： Twelve cats were used in this experiment and 9 of them entered the stage of result analysis. （1） 2 of 10 cats in the iscbemia and reperfusion group died after ischemical reperfusion injury .Resting electreeneephalographie waves did not appear after occlusion of blood vessel in one cat. Seven models were established successfully and the successful rate was 70%. Cats in the sham-operation group all survived. All the cats were paralyzed and in disequilibrium at various degrees after ischemical reperfu- sion injury, and the symptoms were relieved one week later. （2） Most of the primary culture cells were dead after cultured in serum-free medium for two or three days, most of the cells died, only a few of single cells survived in suspension. Most of the cells were splitted into neurosphere with several to several dozens of cells. Cell clump containing several scores to hundreds of cells formed and survived in suspension. The cells in the suspension could adhere and differentiate partially into neurofilament antigen-positive neurons and glial fibrillary acidic protein antigen-positive glia cells in the serum medium. （3）After cells grafting, the signs of palsy and disequilibrium were relieved, but there was not significant difference between experimental side and control side. （4） Many neural stem cells of mouse were detected by immunofluoreseenee in forebrain of cats. one month after transplantation, some of the cells differentiated into neurons and glial cells, some of the cells migrated into brain tissue of cats, the vertical extent of migration was about 0.1 to 0.2 nun, and the distribution of cells was diffusive. The cells which attached to lateral wall of pin holes were round, but the cells which migrated into brain tissue were spindle-shaped. There were not positive cells were detected at hippecampus and distant brain tissue, There was not significant deviation among different individuals. （5） The cells stained with haematoxylin and eosin displayed typical tardus neuron cellular necrosis in pallium and hippocampal CA1. CONCLUSION： Mouse neural stem cells can survive, migrate and differentiate in global brain ischemia models of cats; The experimental model of brain ischemia treated with neural stem cells graft is established successfully.
出处
《中国临床康复》
CSCD
北大核心
2006年第25期27-30,i0002,共5页
Chinese Journal of Clinical Rehabilitation
