嗅成鞘细胞移植治疗脑梗塞大鼠的实验研究

目的探讨嗅成鞘细胞移植在皮层梗塞灶中的治疗价值。方法从成年SD大鼠嗅球取材,差异贴壁法纯化培养,移植时Hoechst33342标记;按双肾双夹方法复制RHRSP模型,RHRSP术后10周,移植组和对照组制作MCAO模型,假手术组不电凝大脑中动脉。选择皮层梗塞10d后的大鼠,在立体定向架上将Hoechst标记的嗅成鞘细胞植入皮层梗塞灶周围,对照治疗组则注入达乐伯克改良培养基,于移植前及移植术后第2周、第6周进行行为、触觉功能检查并从各组随机抽取7只大鼠处死,取大脑,制作冰冻切片用于荧光观察及生长相关蛋白-43及神经中丝免疫组化染色。结果移植后的OECs沿着胼胝体大量向梗塞灶及对侧皮层迁移;移植组和对照组大鼠均出现功能恢复,移植组大鼠运动、感觉功能恢复优于对照组(P<0.05);移植组和对照组都有神经纤维向梗塞中心区生长,移植组的神经纤维数量明显多于对照组(P<0.01);在移植后的第2周、第6周,移植组胼胝体区及梗塞灶相应的对侧皮层区出现GAP-43表达局部升高,其阳性信号值明显高于对照组(P<0.01);梗塞灶周围再生神经纤维的数量与运动功能评分之间存在正相关(r=0.93),P<0.01。结论OECs植入体内可以长时间存活,并有自我迁移能力;OECs移植具有明显促进脑梗塞后神经纤维再生和功能恢复的作用,胼胝体及梗塞灶对侧层的轴突再生也是移植组大鼠功能恢复的可能机制。

Experimental Study of Olfactory Ensheathing Cells Transplantation in Cortical Infarction Rat

Objective To explore the therapeutic effects of olfactory ensheathing cells(OECs) transplantation in cortical infarction rats. Methods OECs were harvested from the olfactory bulb of adult SD rats, the cells were purified using differential time adherent method. OECs were labeled with Hoechst 33342 before transplantation. RHRSP model was replicated by the two-kidney, two-clip method. Ten weeks after RHRSP, MCAO model was replicated. The procedure for the control group was the same as the experimental group except for the coagulation of MCA. At 10 days post-ischemia, Hoechst 33342 labelled OECs cells were transplanted into the cortex surrounding the infarction. In control group, animals only received DMEM medium. Locomotion behaviour and somatosensory score were studied before and 2 to 6 weeks after transplantation. Coronal cryostat sections were also prepared for immunohistochemical staining of GAP-43 and NF. Results OECs were found to migrate from the injection sites along corpus callosum to contralateral cortex and infarction foci. Locomotion behavior and somatosensory deficit were improved. Motor performance and somatosensory score of animals in transplantation group were significantly higher than that of control group in all periods (P<0.05). Nerve fiber regeneration was observed in the core of infarction area in both control and experimental group animals.The number of nerve fibers in animals of transplantation group was higher than the control animals (P<0.01).Immuno-reactivity of GAP-43 in the corpus callosum and specific regions of the contra lateral sensory motor cortex of transplantation group was significantly higher than the control group (P<0.01). There was also a positive correlation between the increase in the number of fiber regeneration and the recovery of locomotion function (r=0.93,P<0.01). Conclusion The capability of OECs to repopulate in the infracted cortex was demonstrated in this study. The capability of OECs to promote the axonal regeneration and the recovery of locomotion and somatosensory function, and also the expression of GAP-43 in corpus callosum and contralateral sensorimotor cortex may explain the functional recovery of animals after transplantation.