【摘要】 目的:比较C17.2神经干细胞和NURR1基因修饰的C17.2神经干细胞移植治疗对脑梗死模型鼠神经功能缺损的改善效果,观察细胞移植后与宿主脑组织的整合、存活、移行及分化情况。方法:实验于2005-06/12在中山大学附属第二医院林百欣实验中心完成。①实验材料:选取清洁级健康SD雄性大鼠78只,随机数字表法分为模型对照组、神经干细胞组、转基因神经干细胞组,26只/组。条件永生化神经干细胞系C17.2由哈佛大学儿童医院Even Synder教授惠赠。pAdeasy-NURR1重组复制缺陷性腺病毒由本实验室成功构建。Dil18荧光染料(Chemicon公司产品)。②实验方法:收集C17.2神经干细胞和pAdeasy-NURR1+C17.2神经干细胞,各分为两管,一管加入Dil18荧光示踪剂。各组大鼠均采用线栓法建立局灶性脑缺血/再灌注损伤模型,3d后进行细胞移植,以江湾Ⅱ型脑立体定向仪进行立体定位,选择缺血半暗区3个位点为移植区:前囟头侧1mm,旁开2mm,深度1.2mm;前囟尾侧3mm,旁开1.5mm,深度1.2mm;前囟尾侧6mm,旁开2mm,深度1.2mm。模型对照组每位点注射单纯培养液2μL;神经干细胞组取6只每位点移植Dil18标记的C17.2神经干细胞悬液2μL,剩余20只每位点移植C17.2神经干细胞悬液2μL;转基因神经干细胞组取6只每位点移植Dil18标记的pAdeasy-NURR1+C17.2神经干细胞悬液2μL,余20只每位点移植pAdeasy-NURR1+C17.2神经干细胞悬液2μL。③实验评估:各组大鼠分别在移植前1d及移植后1周、2周、1个月进行神经功能缺损评分。移植后1周、2周、1个月进行荧光示踪检查及神经元特异烯醇化酶免疫组化检测。移植后6个月行组织学检查。结果:移植后1周,模型对照组死亡1只,神经干细胞组死亡2组,转基因神经干细胞组死亡2只,均淘汰并予以补充。①神经功能缺损评分:移植前1d各组大鼠神经功能缺损评分基本相似(P>0.05)。移植后1周、2周、1个月神经干细胞组、转基因神经干细胞组神经功能缺损评分均明显低于模型对照组(P<0.05);转基因神经干细胞组下降趋势较神经干细胞组更为显著(P<0.05)。②Dil18荧光细胞示踪结果:移植后1周荧光示踪细胞主要在针道内,少数细胞开始移行至周围神经组织。移植后2周,细胞移行至更远距离,并明显向梗死灶方向移行,细胞有突触样结构与周围细胞形成联系。移植后1个月细胞向周围扩散,甚至在远隔部位也可见荧光细胞。③神经元特异烯醇化酶免疫组化检测结果:移植后2周、1个月,转基因神经干细胞组神经元特异烯醇化酶阳性细胞数均明显高于神经干细胞组(P均<0.05)。④细胞组织学检查:移植后6个月,各组大鼠注射针道周围脑组织仍呈正常的层次和结构,未见有明显异型细胞增生。结论:①NURR1基因修饰的C17.2神经干细胞移植治疗局灶性脑梗死能显著改善神经功能缺损,效果优于C17.2神经干细胞。②移植后供体细胞存活并向注射针道周围神经组织移行,促进神经干细胞向神经元方向的分化。更多还原

【Abstract】 AIM: To compare the amelioration of C17.2 neural stem cells (NSCs) and NURR1 plus C17.2 NSCs transplantation on neurologic impairment of the rat models of cerebral infarction. To observe the integration, survival, migration and differentiation of donor cells after transplantation. METHODS: The experiment was conducted at Linbaixin Experimental Center of Second Affiliated Hospital of Sun Yat-sen University from June to December 2005. ① A total of 78 healthy male SD rats of clean grade were selected and randomly divided into model control group (group A), C17.2 neural stem cell transplantation group (group B) and NURR1 plus C17.2 neural stem cell transplantation group (group C) with 26 in each group. Conditionally immortal NSCs line C17.2 was presented by professor Even Synder who worked at Children’s Hospital of Harvard University. The recombinant replication-defective adenovirus pAdeasy-NURR1 has been successfully constructed in this laboratory. Dil18 fluorescent dye was bought from Chemicon Company. ② C17.2 cells and pAdeasy-NURR1 plus C17.2 cells were collected and divided into two tubes, and then the Dil18 fluorescent dye was added into one of them. Rat models of focal cerebral ischemia/reperfusion injury were set up by thread-embolism method. Three days later cell transplantation was performed. Stereotaxis was done with Jianghuang type Ⅱ cerebral stereotaxic apparatus. Three sites were chosen in ischemic periphery area as transplanted sites: anterior fontanel anterior as 1 mm, lateral as 2 mm, ventral as 1.2 mm; anterior fontanel caudal as 3 mm, lateral as 1.5 mm, ventral as 1.2 mm; anterior fontanel caudal as 6 mm, lateral as 2 mm, ventral as 1.2 mm. Group A received 2 μL simple culture fluid at each of the 3 sites. Six rats taken from group B were infused with 2 μL C17.2 NSCs suspension labeled Dil18 fluorescent dye at each sites, and the others (20 rats) were infused with 2 μL C17.2 NSCs suspension at each sites. Six rats taken from group C were infused with 2 μL pAdeasy-NURR1 plus C17.2 NSCs suspension labeled Dil18 fluorescent dye at each sites, and the others (20 rats) were infused with 2 μL pAdeasy-NURR1 plus C17.2 NSCs suspension at each sites. ③ Modified Neurological Severity Score Point (mNSS) was performed 1 day before transplantation and 1 week, 2 weeks and 1 month after the transplantation. Fluorescent-tracking experiment and neuron-specific enolase(NSE) immunohistochemical staining were conducted 1 week, 2 weeks and 1 month after the transplantation. Histological examination was performed 6 months after transplantation. RESULTS: One rat in group A, two in group B and two in group C died 1 week after transplantation. All of them were condemned and supplemented. ①Neurologic impairment score: Neurologic impairment in all groups was similar 1 day before transplantation(P > 0.05). The neurologic impairment score was significantly lower in group B and group C than the group A 1 week, 2 weeks and 1 month after transplantation (P < 0.05). The improvement in the group C was more significant (P < 0.05). ②Result of Dil18 fluorescence cell trace: After transplantation for 1 week, NSCs were mainly in pinhole and a few cells began to migrate to peripheral neural tissues. 2 weeks later, NSCs migrated to further sites, especially significant in the infarcted site, and showed synapse-like structure and connected with peripheral cells. 1 month later, the migration was farer and the fluorescent cells could be seen on remote site. ③Result of NSE immunohistochemical test showed that 2 weeks and 1 month later, the positive rate in group C was significantly higher than that in the group B (P < 0.05). ④Cytohistology examination showed that brain tissues around pinhole still showed normal layer and structure, and no obvious hyperplasia of allotype cells was seen 6 months after transplantation. CONCLUSION: ①The pAdeasy-NURR1 plus C17.2 NSCs transplantation in the treatment of focal infarction can markedly ameliorate neurologic impairment, and the effect is better than that of C17.2 NSCs. ②The primitive and filial generation cells may migrate to the surrounding brain tissue, which can help NSCs differentiate into neurons type.更多还原