白血病抑制因子对转基因SOD1小鼠内源性神经干细胞分化的调控

Regulatory effect of leukaemia inhibitory factor on the differentiation of endogenous neural stem cells in transgenic SOD1 mice

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： After amyotrophic lateral sclerosis, motor neurons have changes in neural degeneration, but the precise mechanism is not clear. Endogenous neural stem cells （ENSCs） are considered to be one of therapeutic methods of treating these kinds of disease in the future. OBJECTIVE： To investigate the effect of leukaemia inhibitory factor on the activation and differentiation of ENSCs in the brainstem of transgenic mice with amyotrophic lateral sclerosis. DESIGN, TIME AND SETTING： The repetitive observation, animal function and cell immunohistochemistry experiment was performed at the Tianjin First Central Hospital from March 2006 to April 2008. Partial experiment was conducted at the Medical College of University of Melbourne, Australia between January and May 2004. MATERIALS： 108 transgenic mice with mutant human superoxide dismutase 1 gene and sourced from B6SJL-TgN （SOD1-G93A） were obtained from Jackson Laboratory, USA, and then randomly assigned into a normal control group, an amyotrophic lateral sclerosis group and a treatment group. Twelve mice, 50% male and 50% female, were selected from each group for this study at 60, 90 and 120 days after delivery. METHODS： Motor function of each mouse was measured at 57 days after delivery to record the dwell time on the Rotarod, once a day. The longest time was 180 s. Mice were obtained at 90 and 120 days after delivery. These mice were treated with drugs at 60 days. Saline supplemented with 25 μg/kg leukaemia inhibitory factor were intraperitoneally infused into mice of the treatment group. Saline of the same volume was injected into mice in the normal control and amyotrophic lateral sclerosis groups, once a day. Corresponding tests were stopped at 60, 90 and 120 days after delivery to isolate mouse brainstem. ENSCs were double labeled using the immunohistochemistry. MAIN OUTCOME MEASURES： Motor function, activation of ENSCs and cell differentiation after activation. RESULTS： At 60 days after delivery, the longest dwell time on the Rotarod was 180 s, and no significant activate ENSCs were detected in the brainstem in each group. At 90 and 120 days, the longest dwell time on the Rotarod was 180 s, but no significant ENSCs were tested in the normal control group. The dwell time on the Rotarod significantly decreased in the amyotrophic lateral sclerosis group and the treatment group, but the reduction was significantly lower in the treatment group than in the amyotrophic lateral sclerosis group （P 〈 0.01）. Significant ENSCs activation was examined in the amyotrophic lateral sclerosis and treatment groups, and the quantity of activated cells were significantly more in the treatment group than in the amyotrophic lateral sclerosis group （P 〈 0.05; P 〈 0.01 ）. Compared with the amyotrophic lateral sclerosis group, the ratio of the differentiation of ENSCs into astrocytes was significantly reduced in the treatment group （P 〈 0.01）; the ratio of the differentiation of ENSCs into neurons and oligodendrocytes was significantly increased （P 〈 0.01 ; P 〈 0.05）. CONCLUSION： Leukaemia inhibitory factor is a neurotrophic factor in up-regulating the expression of ENSCs, switching the differentiation into neurons and oligodendrocytes.