电刺激小脑顶核促进大脑中动脉缺血鼠共移植体中神经干细胞向神经元的分化

Neuronal differentiation of neural stem cells in co-transplanted rats with middle cerebral artery occlusion under electric stimulation of cerebellar fastigial nucleus

目的:向大脑中动脉缺血大鼠植入神经干细胞,探讨电刺激小脑顶核与神经干细胞共移植体对其向神经元分化的影响。方法:实验于2005-08/2006-11在佳木斯大学神经科学研究所完成。①选取同一基因背景大鼠72只,随机数字表法分成3组:神经干细胞移植组、电刺激+神经干细胞组、电刺激+神经干细胞共移植体组,24只/组。②另选取出生24h内的Wistar鼠1只用于神经干细胞的分离,制备单细胞悬液,调整细胞密度为5×108L-1,置于10mg/L的Brdu完全培养液中孵育72h。神经干细胞共移植体由神经干细胞、层粘连蛋白和血管内皮细胞构成,由本实验室提供并鉴定。③各组大鼠于造模前1d麻醉后行小脑顶核刺激,以前囟为零点、正中线向后11.6mm、正中线向右1.2mm。给予直角方波脉冲刺激,电流强度为50μA,频率80Hz,时程1h。④各组大鼠均采用线栓法建立局灶性脑缺血动物模型。造模1d后,分别将培养的神经干细胞及共移植体细胞浓度调整为2×1010 L-1,于缺血纹状体侧缺血半暗带区垂直进针,进入5.0mm后缓慢推入细胞悬液,神经干细胞移植组、电刺激+神经干细胞组移植神经干细胞悬液10μL,电刺激+神经干细胞共移植体组移植神经干细胞共移植体悬液10μL,移植速度为0.5μL/min,留针10min后缓慢拔针。⑤各组分别于移植后3,7,14,60d观察神经干细胞移入脑内后分化成神经元的情况。胞核呈绿色荧光、胞质呈红色荧光的为Brdu-神经元特异性烯醇化酶双阳性细胞,代表移植入的神经干细胞所分化的神经元。结果:72只同一基因背景大鼠均进入结果分析。①与神经干细胞移植组比较,移植后7,14,60d电刺激+神经干细胞组、电刺激+神经干细胞共移植体组Brdu-神经元特异性烯醇化酶双阳性细胞百分率均明显升高(F=12.44～25.18,P均<0.05)。移植后3,7,14,60d电刺激+神经干细胞共移植体组Brdu-神经元特异性烯醇化酶双阳性细胞百分率均明显高于电刺激+神经干细胞组(F=8.19～25.18,P均<0.05)。②随移植时间的延长,各组Brdu-神经元特异性烯醇化酶双阳性细胞形态均逐渐增大,至移植后60d时细胞周边可见突起。结论:大脑中动脉缺血模型鼠移植入神经干细胞后,电刺激小脑顶核能够对神经干细胞向神经元的分化产生促进作用,且共移植体与电刺激小脑顶核具有协同效应。

AIM： To explore the effects of electric stimulation of carebellar fastigial nucleus on the differentiation of neurons from neural stem cells （NSCs）, which are co-transplanted to middle cerebral artery occlusion （MCAO） rats. METHODS： The experiment was finished from August 2005 to November 2006 in the Institute of Neuroscience of Jiamusi University. ①Seventy-two rats with the same genetic background were randomly divided into 3 groups： NSCs group, fastigial nucleus stimulation （FNS） and NSCs group, FNS and NSCs co-transplant group, each contained 24 rats. ②One neonatal Wistar mice were used to isolate NSCs and prepare the mononuclear suspension at the density of 5×10^8 L^-1. NSCs were cultured in 10 mg/L Brdu complete medium for 72 hours. The co-transplant was composed of NSCs isolated from hippocampi, vascular endothelial calls isolated from cerebra cortex and laminin, which were all offered and identified by this laboratory. ③The rats were stimulated by FNS 1 hour before MCAO operation in the size of posterior 11.6 mm and right 1.2 mm of median line, taking anterior fontanelle as null point. Square pulse stimulation parameter： current intensity 50 μA, frequency 80 Hz, time course 1 hour. ④Focal cerebral ischemia model was prepared using filament method, one hour later, NSCs and co-transplant at the density of 2×10^10 L^-1 were separately transplanted to the ischemic penumbra of MCAO rats. Then cell suspension was gradually given at the velocity of 0.5 μL/min and remained for 10 minutes, 10 μL NSC suspension in NSCs group, FNS and NSCs group, while 10 μL NSC co-transplant suspension in FNS and NSCs transplant group. ⑤The differentiation of NSCs into neurons were observed through immunofluorescance staining at the 3^rd day, 7^th day, 14^th day, 60th day after transplantation. The Brdu-neuronspecific enolase （NSE）-positive cells indicated the neurons, in which green dots represented nucleus and red dots represented cytoplasm. RESULTS： All 72 rats were included in the result analysis. ①At the 7^th day, 14^th day, 60^th day of transplantation, the percentage of Brdu-NSE-positive cells were obviously increased in the FNS and NSCs group, FNS and NSCs co-transplant group compared with NSCs group （F=12.44-25.18, P 〈 0.05）. At the 3^rd day, 7th day, 14^th day, 60^th day of transplantation, the percentage of Brdu-NSE-positive cells were obviously higher in FNS and NSCs co-transplant group than in FNS and NSCs group （F =8.19-25.18, P 〈 0.05）. ② With the prolong of transplantation time, the Brdu-NSE-positive cells became larger, and appeared prominence at the 60^th day. CONCLUSION： Electric stimulation of cerebellar fastigiul nuclus improves the differentiation to neurons of NSCs transplanted to MCAO rats, co-transplant and FNS improve the differentiation in coordination.