The Role of Exogenous Neural Stem Cells Transplantation in Cerebral Ischemic Stroke
The Role of Exogenous Neural Stem Cells Transplantation in Cerebral Ischemic Stroke
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DOI：
10.1166/jbn.2014.2018
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发表时间：
2014-11-01
影响因子：
2.9
通讯作者：
Gu, Ning
中科院分区：
文献类型：
--
作者：
Chen, Lukui;Qiu, Rong;Gu, Ning
目的观察大鼠纹状体及室管膜下区神经干细胞移植对局灶性脑缺血再灌注损伤的影响。从孕14天的胎鼠中提取海马。采用悬浮培养法分离培养大鼠神经干细胞。采用结扎大鼠左侧大脑中动脉的方法制备大鼠左侧脑缺血再灌注模型。采用Zea Longa五级量表评估神经系统体征，以1、2、3分判定大鼠模型建立成功。造模成功后24 h，将神经干细胞立体定向注射入大鼠左侧纹状体。将大鼠随机分为正常组、假手术组、缺血组、PBS移植组和NSCs移植组，分别于第3、7、14天进行观察。采用7分评价标准评价缺血相关神经功能缺损。采用足错法对所有大鼠的前肢损伤进行评价。TTC染色观察脑梗死灶大小变化，尼氏染色观察梗死区细胞形态结构变化。TUNEL法检测细胞凋亡和凋亡阳性细胞计数。采用BrdU/NeuN和BrdU/GFAP荧光双标记法观察纹状体和室管膜下区（SVZ）双标记阳性细胞的表达，并计数阳性细胞数。不同处理组的结果显示，缺血组、PBS移植组、NSCs移植组均出现不同程度的右侧偏瘫。与前两组相比，神经干细胞移植组偏瘫发生率最低。TTC染色显示神经干细胞移植组大鼠梗死体积小于PBS移植组。尼氏染色显示缺血组和PBS移植组均可见大面积神经元坏死和凋亡，损伤主要集中在纹状体。神经干细胞移植组神经细胞变性和损伤明显减轻。Tunel法检测结果显示，NSCs移植组各时间点凋亡阳性细胞数均少于PBS移植组。免疫荧光双标显示，SVZ内内源性神经干细胞从第3天开始增殖，第7天达到高峰，第14天明显减少。神经干细胞移植组BrdU/NeuN数量较PBS移植组明显增加（P < 0.05）。神经干细胞移植组BrdU/GFAP表达较PBS移植组明显减少（P < 0.05）。PBS移植组纹状体BrdU/GFAP阳性细胞数明显多于NSCs移植组。神经干细胞移植可明显改善脑缺血大鼠的神经功能缺损和协调能力。因此，神经干细胞移植可能促进内源性神经干细胞向神经元分化，减少其向胶质细胞分化，并可能改变纹状体缺血微环境，抑制胶质细胞的增殖。
To observe the effects of neural stem cells (NSCs) transplantation in rats' striatum and subventricular zone (SVZ) in rat models of focal cerebral ischemia and reperfusion. Hippocampus was extracted from fetal rats with 14 days of gestation. Suspension culture was used to isolate and culture the rat's NSCs. A cerebral ischemia and reperfusion rat's model was made on the left side of the brain through occlusion of the left middle cerebral artery. Neurological signs were assessed by Zea Longa's five-grade scale, with scores 1, 2, and 3 used to determine the successful establishment of the rat's model. The NSCs were stereotaxically injected into the left striatum 24 hours after the successful rat's model was built. Rats were then randomly divided into 5 groups, namely, normal group, sham operation group, ischemia group, PBS transplantation group, and NSCs transplantation group, each of which was observed on day 3, day 7, and day 14. The ischemia-related neurological deficits were assessed by using a 7-point evaluation criterion. Forelimb injuries were evaluated in all rats using the foot-fault approach. Infarct size changes were observed through TTC staining and cell morphology and structure in the infarct region were investigated by Nissl staining. Apoptosis and apoptosis-positive cell counts were studied by Tunel assay. Expressions of double-labeling positive cells in the striatum and subventricular zone (SVZ) were observed by BrdU/NeuN and BrdU/GFAP fluorescent double-labeling method and the number of positive cells in the striatum and SVZ was counted. Results from the differently treated groups showed that right hemiplegia occurred in the ischemia group, PBS transplantation group, and NSCs transplantation group in varying degrees. Compared with the former two groups, there was least hemiplegia in the NSCs transplantation group. The TTC staining assay showed that rats in the NSCs transplantation group had smaller infarct volume than those from the PBS transplantation group. The Nissl dyeing showed that there was a large area of neuronal necrosis and apoptosis in the ischemia and PBS transplantation groups, and damage was mainly focused in the striatum. Degeneration and damage of nerve cells were significantly reduced in the NSCs transplantation group. The Tunel assay showed that the number of apoptosis-positive cells in the NSCs transplantation group was less than that in the PBS transplantation group at each time point. Double immunofluorescent labeling showed that the proliferation of endogenous neural stem cells began at the third day, reaching the peak at the 7th day, and was significantly reduced at the 14th day in the SVZ. The number of BrdU/NeuN increased significantly in the NSCs transplantation group compared to that in the PBS transplantation group (P < 0.05). The number of BrdU/GFAP decreased significantly in the NSCs transplantation group compared to that of PBS transplantation group (P < 0.05). The number of BrdU/GFAP-positive cells in the striatum was observed to be much more in the PBS transplantation group than in the NSCs transplantation group. Both neurological deficits and coordination capacity of rats with cerebral ischemia were significantly improved via transplantation of the neural stem cells. In conclusion, transplantation of neural stem cells can therefore possibly promote the differentiation of endogenous NSCs into neurons and reduce their differentiation towards glial cells.Transplantation of the neural stem cells may also change the ischemic microenvironment of striatum, possibly inhibiting the proliferation of glial cells.