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目的观察人脐血干细胞(human umbilical cord blood stem cells,HUCBSCs)在局灶性脑缺血大鼠体内的迁移与分化,探讨HUCBSCs移植对缺血性脑损伤大鼠神经功能恢复可能的作用机制。方法采用线栓法制备大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)再灌注模型,将造模成功的20只大鼠随机分为2组:移植组15只,造模成功后24 h,经尾静脉移植1 ml(2×106个)DAPI/CM-Dil荧光染料标记的HUCBSCs;模型组5只,造模成功后24 h,经尾静脉移植等量生理盐水。分别于移植前及移植后3、7、15 d进行神经功能缺损评分(neurological severity score,NSS),并取脑组织,制备冰冻切片,HE染色观察脑组织形态,荧光显微镜观察HUCBSCs在脑组织中的迁移和分布,免疫荧光法检测脑组织中巢蛋白(Nestin)和神经元特异性烯醇化酶(neuron specific enolase,NSE)的表达。结果移植组大鼠HUCBSCs移植后7 d起,其改良的NSS(m NSS)显著低于对照组(P<0.05)。移植后15 d,大鼠脑组织病理改变明显减轻。移植后3 d起,可见病灶及其周围部位有阳性细胞存在,随着时间的推移,脑缺血病灶部位的阳性细胞增多;移植后7 d,迁移至病灶部位的细胞数增加;移植后15 d,迁移至病灶部位的细胞数量达观察期内的高峰,各时间点阳性细胞数差异有统计学意义(P<0.05)。移植后15 d,大鼠脑组织中CM-Di I/NSE-FITC和CM-Di I/Nestin-FITC双阳性细胞率分别为85.2%和81.6%。结论 HUCBSCs经静脉移植后,能在大鼠体内存活,并向损伤部位迁移,具有向神经元细胞方向分化的潜能,对脑缺血大鼠的神经功能恢复具有促进作用。
Objective To observe the migration and differentiation of human umbilical cord blood stem cells (HUCBSCs) in focal cerebral ischemia rats, and to explore the possible mechanism of HUCBSCs transplantation on neurological function recovery in rats with ischemic brain injury. Methods The rat model of middle cerebral artery occlusion (MCAO) reperfusion was established by thread occlusion. Twenty rats with successful model were randomly divided into 2 groups: 15 rats in transplantation group, 24 h after successful model establishment , 1 ml (2 × 106) DAPI / CM-Dil fluorescent labeled HUCBSCs were transplanted into the caudal vein. In the model group, 5 rats were injected with the same amount of normal saline via caudal vein 24 h after the successful model establishment. Neurological severity scores (NSS) were taken before and at 3, 7 and 15 days after transplantation. Frozen sections were prepared and stained with HE. The morphological changes of brain were observed by HE staining. HUCBSCs were observed under light microscope The migration and distribution of Nestin and neuron specific enolase (NSE) in brain tissue were detected by immunofluorescence. Results The improved NSS (m NSS) of HUCBSCs transplantation group was significantly lower than that of the control group (P <0.05) at 7 days after transplantation. Fifteen days after transplantation, the pathological changes in rat brain tissue were relieved. At 3 days after transplantation, there were positive cells in the lesion and its surrounding areas. As time went by, the number of positive cells in focal cerebral ischemia increased. At 7 days after transplantation, the number of cells migrated to the lesion increased. After transplantation, d, the number of cells migrating to the lesion reached the peak during the observation period, the number of positive cells at each time point had statistical significance (P <0.05). Fifteen days after transplantation, the positive rates of CM-Di I / NSE-FITC and CM-Di I / Nestin-FITC double positive cells in rat brain were 85.2% and 81.6%, respectively. Conclusion After transplanted into the vein, HUCBSCs can survive in the rat body and migrate to the injured site, have the potential of differentiating into neuronal cells, and promote the recovery of neurological function in rats with cerebral ischemia.