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背景:肌萎缩侧索硬化后运动神经元存在神经变性改变,但其确切机制不明,内源性神经干细胞被认为是未来能有效解决此病的治疗方法之一。目的:探讨白血病抑制因子对肌萎缩侧索硬化转基因小鼠脑干内源性神经干细胞的激活及分化方向的影响。设计、时间及地点:重复观察测量,动物功能学与细胞免疫组化学实验,主要于2006-03/2008-04在天津市第一中心医院完成,部分实验于2004-01/05在澳大利亚墨尔本大学医学院完成。材料:由美国Jackson实验室提供的起源于B6SJL-TgN(SOD1-G93A)、表达突变人类超氧化物歧化酶1基因的转基因小鼠108只,随机均分为正常对照组、肌萎缩侧索硬化组、治疗组,每组动物分别在出生后60,90,120d各取12只用于实验,雌雄各半。方法:各组小鼠均于出生后57d起开始进行运动功能测试,记录小鼠在Rotarod上的停留时间,最大测定时间为180s,每天测试1次,直到实验结束。于出生后90,120d取材的小鼠,从出生后60d开始接受药物干预,治疗组腹腔内注射含25μg/kg白血病抑制因子的生理盐水,正常对照组与肌萎缩侧索硬化组等量注射单纯的生理盐水,1次/d。分别于出生后60,90,120d终止相应实验,分离小鼠脑干,采用双重免疫荧光组化法标记内源性神经干细胞。主要观察指标:运动功能,内源性神经干细胞的激活,激活后细胞分化方向。结果:出生后60d,各组动物均能在Rotarod上停留达到最大测试时间180s,脑干均未发现明显的内源性神经干细胞激活。出生后90,120d,正常对照组仍能达到Rotarod最大测试时间180s,仍没有明显的内源性神经干细胞激活;肌萎缩侧索硬化组、治疗组小鼠在Rotarod上的停留时间均明显降低,但后者降低程度明显小于前者(P<0.01);肌萎缩侧索硬化组、治疗组均出现明显的内源性神经干细胞激活,且后者出现激活的细胞数量明显高于前者(P<0.05;P<0.01)。出生120d时与肌萎缩侧索硬化组比较,治疗组内源性神经干细胞分化为星形胶质细胞的比例明显下降(P<0.01),分化成神经元及少突胶质细胞的比例明显上升(P<0.01;P<0.05)。结论:白血病抑制因子是一种能提升内源性神经干细胞的激活、调控其朝向神经元及少突胶质细胞分化的神经营养因子。
BACKGROUND: There is neurodegeneration in motoneurons after amyotrophic lateral sclerosis, but its exact mechanism is unknown. Endogenous neural stem cells are considered as one of the effective treatments for this disease in the future. Objective: To investigate the effect of leukemia inhibitory factor on activation and differentiation of endogenous neural stem cells in brain stem of amyotrophic lateral sclerosis mice. DESIGN, TIME AND SETTING: The experiment of repeated observation, animal function and Cellular Immunohistochemistry was performed at Tianjin No.1 Central Hospital from March 2006 to April 2008 with some experiments performed at the University of Melbourne, Australia Medical school completed. MATERIALS: A total of 108 transgenic mice expressing the mutant human superoxide dismustase 1 gene from B6SJL-TgN (SOD1-G93A) provided by Jackson Laboratory were randomly divided into normal control group, amyotrophic lateral sclerosis Group, treatment group, each group of animals after 60, 90, 120d each take 12 for experiment, male and female. Methods: The mice in each group started to exercise function test at 57 days after birth. The retention time of mice on Rotarod was recorded. The maximum measurement time was 180 seconds and the test was performed once daily until the end of the experiment. At 90 and 120 days after birth, mice were sacrificed 60 days after birth to receive drug intervention. The treatment group received intraperitoneal injection of saline containing 25 μg / kg of leukemia inhibitory factor. The normal control group and amyotrophic lateral sclerosis group were injected with pure Saline, 1 time / d. The corresponding experiments were terminated 60, 90 and 120 days after birth, respectively. The brainstem of mice was isolated and endogenous neural stem cells were labeled by double immunofluorescence staining. MAIN OUTCOME MEASURES: Motor function, activation of endogenous neural stem cells, and direction of cell differentiation after activation. RESULTS: At 60 days after birth, all the animals in each group could stay on Rotarod for 180s, and no obvious activation of endogenous neural stem cells was found in the brain stem. 90,120 days after birth, the control group can still reach the maximum test time of Rotarod 180s, still no significant endogenous neural stem cell activation; amylase lateral sclerosis group, the treatment group mice Rotarod residence time were significantly reduced, but (P <0.01). Amyotrophic lateral sclerosis group and treatment group showed significant activation of endogenous neural stem cells, and the number of activated cells in the latter group was significantly higher than the former (P <0.05; P <0.01). Compared with amyotrophic lateral sclerosis group, the percentage of endogenous neural stem cells differentiated into astrocytes in the treatment group decreased significantly (P <0.01) at 120 days of birth, and the percentage of differentiated into neurons and oligodendrocytes increased significantly (P <0.01; P <0.05). CONCLUSION: Leukemia inhibitory factor is a neurotrophic factor that enhances the activation of endogenous neural stem cells and regulates its differentiation towards neurons and oligodendrocytes.