摘要
背景:神经炎症是影响肌萎缩侧索硬化进展的重要因素,体外研究显示神经干细胞有一定的抗炎作用,但在肌萎缩侧索硬化鼠体内能否抗炎,其机制及最佳移植途径尚不清楚。目的:研究不同途径移植神经干细胞对肌萎缩侧索硬化模型G93A-SOD1小鼠的抗炎作用及机制。方法:分离、鉴定神经干细胞后用绿色荧光蛋白转染;将78只70日龄G93A-SOD1小鼠分为脑室内注射组、尾静脉注射组、对照组,适应性喂养后,于84日龄经侧脑室移植入5×10^(5) 个绿色荧光蛋白转染神经干细胞,经尾静脉注射1×106个绿色荧光蛋白转染神经干细胞;从85日龄开始,每周进行1次改良Wrathall运动评分、旋转实验来评价运动功能,记录起病时间、病程时间及生存时间;10^(5) 日龄时,采用免疫荧光检测绿色荧光蛋白转染的神经干细胞存活、迁移、分化情况,采用Nissl染色检测运动神经元数量、Western blot检测ChAT的表达、免疫荧光检测脊髓前角运动神经元中NeuN的表达,采用ELISA和RT-PCT检测脑脊液中肿瘤坏死因子α、白细胞介素1β、白细胞介素6、转化生长因子β的蛋白和mRNA水平,采用Western blot检测脊髓组织中iNOS、CD206及NF-κB通路蛋白表达,采用苏木精-伊红染色检测腓肠肌病理变化。结果与结论:①提取的神经干细胞生长、分化良好,可被绿色荧光蛋白转染;②起病后超早期,侧脑室途径更利于神经干细胞进入中枢神经系统,降低肿瘤坏死因子α、白细胞介素1β、白细胞介素6并提升转化生长因子β水平,降低iNOS并提升CD206表达水平,抑制NF-κB通路激活,减缓运动功能损害进展,减轻骨骼肌病理损害程度;③起病后超早期,2种途径移植神经干细胞保护脊髓前角运动神经元、延长小鼠病程时间及生存时间的能力均有限;④结果表明,在肌萎缩侧索硬化的起病后超早期,侧脑室可能是神经干细胞更好的移植途径,具有良好的抗�
BACKGROUND:Neuroinflammation affects the progression of amyotrophic lateral sclerosis.In vitro studies have shown that neural stem cells have antiinflammatory effect,but whether they can be anti-inflammatory in amyotrophic lateral sclerosis mouse,and the mechanism and optimal transplantation route are still unclear.OBJECTIVE:To investigate the anti-inflammatory effect and mechanism of neural stem cell transplantation in different ways on amyotrophic lateral sclerosis model G93A-SOD1 mice.METHODS:Neural stem cells were isolated and identified,transfected with green fluorescent protein.Totally 78 G93A-SOD1 mice aged 70 days were divided into lateral ventricle group,tail vein group,and control group.After adaptive feeding,5×10^(5) green fluorescent protein-transfected neural stem cells were transplanted into the lateral ventricle,and 1×106 green fluorescent protein-transfected neural stem cells were injected through the tail vein at the age of 84 days.From the age of 85 days,modified Wrathall motor score and rotation test were performed every week to evaluate motor function.Onset time,disease duration and survival time were recorded.At the age of 10^(5) days,immunofluorescence method was conducted to detect survival,migration and differentiation of neural stem cells transfected with green fluorescent protein.The number of motor neurons was measured by Nissl staining.The expression of ChAT was detected by western blot assay.Immunofluorescence was used to detect the expression of NeuN in spinal cord anterior horn motor neurons.The protein and mRNA levels of tumor necrosis factor alpha,interleukin 1 beta,interleukin 6 and transforming growth factor beta in cerebrospinal fluid were detected by ELISA and RT-PCT.Western blot assay was used to detect the expression of iNOS,CD206 and NF-κB pathway proteins in the spinal cord.The pathological changes of gastrocnemius muscle were detected by hematoxylin-eosin staining.RESULTS AND CONCLUSION:(1)The extracted neural stem cells grew and differentiated well and could be transf
作者
林彦琛
李晶晶
陆广旭
代二庆
陈静
钟士江
Lin Yanchen;Li Jingjing;Lu Guangxu;Dai Erqing;Chen Jing;Zhong Shijiang(Department of Rehabilitation Medicine,Characteristic Medical Center of the Armed Police,Tianjin 300162,China;Department of Neurology,Characteristic Medical Center of the Armed Police,Tianjin 300162,China;Department of Pharmacy,Tianjin Fourth Central Hospital,Tianjin 314000,China;The Second Mobile Corps Hospital of the Armed Police,Wuxi 214000,Jiangsu Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第15期2363-2370,共8页
Chinese Journal of Tissue Engineering Research
基金
天津市自然科学基金项目(13JCBJC24100),项目负责人:钟士江
天津市卫生行业重点攻关项目(16KG136),项目负责人:钟士江。
