G-CSF诱导APP转基因小鼠神经再生及对认知功能改善作用的研究
|
摘要
研究背景
阿尔茨海默病(Alzheimer's disease, AD)是一种起病隐袭的中枢神经系统退行性疾病,以记忆力进行性减退、认知功能障碍、行为活动异常为主要临床特征。其患病率约占老年痴呆病例的一半以上,随着人口老龄化的加重,阿尔茨海默病的发病率逐年增高。AD严重影响患者的工作能力和生活质量,也为家庭和社会带来沉重的负担。鉴于AD的发病机制尚未阐明,目前尚无理想的预防和治疗方法。
AD的主要病理特征是在患者大脑皮质、海马、某些皮质下神经核出现老年斑(senile plaque, SP)和神经原纤维缠结(neurofibrillary tangle, NFT),此外还有神经元缺失、星形细胞增生样反应、神经元突触异常等病理改变。AD的核心症状是认知功能进行性下降,谷氨酸能及胆碱能神经元丢失程度与患者智能衰退关系密切。因此,减少神经元丢失或增加神经再生是AD治疗的重要方向。
粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF)是由单核细胞、成纤维细胞和内皮细胞产生的一种造血生长因子,能与细胞表面的特定受体结合,促使中性粒细胞系造血祖细胞生长和分化,保护中性粒细胞避免凋亡并加强它们的功能,G-CSF被广泛地应用于治疗由各种原因引起的粒细胞减少症。最近多项研究显示其对中枢神经系统也有多重效应,可促进骨髓源性干细胞向受损脑区迁移,抗细胞凋亡,促进神经细胞再生。神经干细胞存在大脑特定区域,特别是脑室下区(管膜)、嗅球和海马,这些区域均可表达G-CSF及G-CSFR。G-CSF具有神经保护作用,能促进组织形态修复和神经功能康复。近年来,对成年动物脑缺血模型的研究表明,应用G-CSF是一种有效治疗方法,Kawada等研究发现,缺血性脑卒中动物模型经G-CSF治疗,能通过动员骨髓干细胞而改善卒中后脑功能受损症状。国外应用G-CSF治疗成人缺血性脑卒中已进入Ⅰ、Ⅱ期临床观察阶段。
根据以往研究推测,G-CSF可能通过促进神经再生治疗AD,改善AD患者的认知功能。本研究将G-CSF应用于APPV717I转基因AD模型小鼠,以探讨G-CSF能否促进神经再生和改善转基因小鼠认知能力。本研究共分三部分,如下：
第一部分G-CSF对阿尔茨海默病小鼠认知功能的影响
目的对G-CSF治疗前后AD模型小鼠、对照组和野生小鼠进行生物行为学研究,利用水迷宫模型来测试各组小鼠学习记忆能力,探讨G-CSF能否改善AD小鼠模型的认知能力。
方法1.将10月龄APPV717I转基因AD模型小鼠和野生小鼠分为对照组、G-CSF治疗组和野生组。G-CSF治疗组：连续7d皮下注射G-CSF(50μg/kg·d)。对照组、野生组,连续7d皮下注射PBS。按照G-CSF治疗完成后的天数,各组小鼠又分为三个亚组：7d组,14d组,28d组。
2.各组小鼠于注射前后分别进行水迷宫定位航行试验。
3.利用统计软件对G-CSF治疗前后的AD模型小鼠及野生小鼠的水迷宫实验数据进行统计分析。观察G-CSF对AD模型小鼠认知功能的影响。
结果各组小鼠Morris水迷宫测试结果：AD模型组逃避潜伏期和游泳距离较野生组明显延长(P<0.01),G-CSF治疗组显著缩短潜伏期和游泳距离,和野生组水平相当。同组大鼠,随着训练时段的增加,G-CSF治疗组的潜伏期和游泳距离越来越短(P<0.01)。各组小鼠游泳速度没有明显差异。可以排除因个体差异引起的不同。
结论G-CSF治疗后阿尔茨海默病小鼠认知功能改善。
第二部分G-CSF对阿尔兹海默病小鼠CD 34+/ CD 45+细胞比例的影响
目的应用流式细胞学的方法观测G-CSF治疗前后APP转基因模型小鼠外周血中CD 34+/CD 45+细胞比例,探讨G-CSF对阿尔茨海默病模型小鼠骨髓造血干细胞的影响。
方法1.将10月龄APPV7171转基因AD模型小鼠分为对照组,G-CSF治疗组。G-CSF治疗组：连续7d皮下注射G-CSF(50μg/kg·d)。对照组：连续7d皮下注射PBS。
2.给药第14 d Morris水迷宫测试后从小鼠眶后静脉丛取外周血0.5 ml,进行流式细胞学测试,观察2组小鼠外周血中CD 34+/CD 45+细胞比例。
结果治疗组小鼠外周血CD 34+/CD45+细胞数较对照组明显增加(P<0.01),约为对照组3倍。
结论1.G-CSF能提高AD模型小鼠外周血中CD 34+/CD 45+细胞的比例。
2.G-CSF能够动员AD模型小鼠CD34+的骨髓造血干细胞(hematopoietic stem cells,HSC)增殖并进入外周血。
第三部分G-CSF对阿尔兹海默病小鼠脑内神经元再生的影响
目的对G-CSF治疗前后AD模型小鼠脑组织切片进行免疫组化及免疫荧光双标检测,通过CD 34+免疫荧光染色、Nestin+/BrdU+、MAP-2+/BrdU+免疫荧光双重标记,观察G-CSF能否影响AD小鼠模型脑内的神经再生
方法1G-CSF治疗组：连续7d皮下注射G-CSF(50μg/kg·d)。对照组：连续7d皮下注射PBS。给药同时,两组动物给予BrdU(50 mg/kg·d)腹腔注射,连续10d。
2.在给药后14d将两组小鼠多聚甲醛灌注取脑,冰冻切片,CD 34+免疫荧光染色、Nestin+/BrdU+、MAP-2+/BrdU+免疫荧光双重标记。
结果1.治疗组在给药后第14d时脑内的皮质、嗅球及海马区可检测到红色的CD 34+细胞。
2.在两组小鼠脑内的皮质、嗅球及海马区均检测到BrdU+细胞,治疗组较对照组显著增加(p<0.05)。
3.应用免疫荧光双标染色,在皮质、嗅球及海马区均可见Nestin+/BrdU+、MAP-2+/BrdU+双阳性细胞,较对照组明显增加(p<0.05)。
结论1.G-CSF可诱导AD模型小鼠骨髓造血干细胞向脑内迁移。
2.G-CSF能明显促进AD模型小鼠脑内细胞增殖。
3.G-CSF可诱导AD模型小鼠脑中神经干细胞增殖并向神经元方向分化。
研究意义
本研究结果表明G-CSF皮下注射可明显改善APP转基因小鼠的认知功能,其作用机制为动员外周骨髓造血干细胞增殖、分化,并向脑内定向迁移；同时诱导脑内神经干细胞增殖、向神经元分化,从而替代丢失的神经元,修复病灶,为G-CSF治疗阿尔茨海默病提供了新的实验依据。
Background
Alzheimer's disease (AD) is an insidious-onset neurodegenetative disease of central nervous system (CNS), characterized by progressive memory loss, cognitive dysfunction and behavior disorder. AD occupies more than half of aging dysmnesia. With the increasing proportion of the elderly in the population, its incidence increases year by year. The working ability and life quality of the patients are damaged significantly and this disease places a heavy burden on the family and society. With its pathogenesis still veiled, AD can not be prevented and treated effectively.
The main pathological changes of AD include senile plaque (SP) and neurofibrillary tangle (NFT) in the cerebral cortex, hippocampus and subcortical nucleus, as well as neuron loss, astrocytosis, and the abnormality of neurons and synapsis. The outstanding symptom of AD is progressive cognitive dysfunction, which is closely related to the loss of glutamatergic and cholinergic neurons. Therefore, reducing neuron loss and increasing neuroregeneration is an important trend of AD treatment.
Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor produced by monocyte, fibroblast and endotheliocyte, can bind with specific receptors on the cellular surface, promote the proliferation and differentiation of granulocyte hemopoietic progenitor, protect the neutrophils from apoptosis and intensify their function. G-CSF has been widely used in the treatment of granulocytopenia caused by various reasons. Several recent researches indicated that G-CSF could exert multiple effects on CNS, including inducing marrow-derived stem cells to migrate to the impaired brain area, anti-apoptosis, promoting neurogenesis. Neural stem cells exist in certain areas within the brain, especially subventricular zone area (ependymal), olfactory bulb and hippocampus, where G-CSF and G-CSFR are expressed. G-CSF plays such a neuroprotective role that it promotes the recovery of nervous tissue both functionally and morphologically. Recently, researches on adult animal models of cerebral ischemia showed that G-CSF is an effective drug. Kawada and his team found that the G-CSF treatment for the animal model of ischemic stroke could improve the recovery of the ischemic brain function by mobilizing bone marrow stem cells. The treatment of G-CSF for adult ischemic stroke has entered PhaseⅠandⅡclinical trials abroad.
Based on previous researches, G-CSF might be able to improve cognitive function by promoting neurogenesis. In this research, G-CSF was administrated to APPV7171 transgenic mouse model of AD in order to determine whether G-CSF could induce neuroregeneration and improve the cognition. This research consisted of 3 parts, which would be described as follows.
Part 1. The influence of G-CSF on the cognition of AD mice
Purpose:To study the changes of biological behavior of AD mice after the administration of G-CSF, Morris water maze was used to test their study and memory functions, which was in order to discuss whether G-CSF treatment could improve the cognition of AD mice.
Method:1.Divide 10-month old APPV7171 transgenic AD mice and wild type group randomly into the G-CSF group, the control group and the wild type group. G-CSF group:Subcutaneous injection of G-CSF (50μg/kg·d) for 7 days in succession. Control group and wild type group:Subcutaneous injection of PBS for 7 days in succession. According to the time after the G-CSF treatment, each group was divided into 3 sub-groups:7-day subgroup,14-day subgroup and 28-day subgroup.
2. Each group of mice was under Water maze orientation navigation experiments before and after injection.
3. Analyze the data of Water maze orientation navigation experiments with statistics software. Observe the influence that G-CSF exerts on the AD mice.
Results:Results of Water maze orientation navigation experiments of each group: Comparing to the wild type group, the escape response latency and swimming distance of the AD mice were significantly prolonged; those data of the G-CSF group were shortened significantly and similar with the wild type group. Among mice of the same group, the shortening of the escape response latency and swimming distance increased as the training time extended (P<0.01). There was no significant difference between the swimming speeds of each group. Difference caused by individual variation could be excluded.
Conclusions:The cognitive function of AD mice was improved after the G-CSF treatment.
Part 2 The influence of G-CSF on the CD 34+/CD 45+ratio of AD mice
Purpose:With flow cytometry, we investigated the difference of CD 34+/CD 45+ ratio between the G-CSF group and the control group, which aimed to discuss the influence of G-CSF on the hematopoietic stem cells of AD mice.
Method:1. Divide 10-month old APPV7171 transgenic AD mice randomly into the G-CSF group and the control group. G-CSF group:Subcutaneous injection of G-CSF(50μg/kg·d) for 7 days in succession. Control group:Subcutaneous injection of PBS for 7 days in succession.
2. On the 14th day after the injection, take 0.5ml of peripheral blood from postorbital vein plexus of mice after Morris water maze test and observe the peripheral CD 34+/CD 45+ ration with flow cytometry.
Results:The peripheral CD 34+/CD45+ ratio of G-CSF group was significantly increased compared to the control group (P<0.01), which was about 3 times as large as that of the control group.
Conclusions:1. G-CSF elevated CD 34+/CD 45+ ratio in the peripheral blood of AD mice.
2. G-CSF could promote the proliferation of CD 34+ hematopoietic stem cell (HSC) and mobilize those cells into peripheral blood.
Part 3 The influence of G-CSF on the neurogenesis of AD mice
Purpose:By performing the immunohistochemical and immunofluorescence double-labeled detection tests on the G-CSF group and the control group (immunofluorescence staining of CD 34+, immunofluorescence double-labeling of Nestin+/BrdU+ and MAP-2+/BrdU+), discuss whether G-CSF could influence the neurogenesis.
Method:1. G-CSF group:Subcutaneous injection of G-CSF (50μg/kg·d) for 7 days in succession. Control group:Subcutaneous injection of PBS for 7 days in succession. During the period, both groups were given intraperitoneal injection of BrdU (50 mg/kg·d) for 10 days in succession
2. On the 14th day after the injection, remove the mice brain of both groups by paraformaldehyde perfusion, and frozen on dry ice for the specimen. Then conduct the immunofluorescence staining of CD34+, and immunofluorescence double-labeling of Nestin+/BrdU+, MAP-2+/BrdU+.
Results:1. On the 14th day after the injection, red-stained CD34+ cells were found in subventricular zone area, olfactory bulb and hippocampus of the G-CSF group.
2. BrdU+ cells could be detected in subventricular zone area, olfactory bulb and hippocampus of both groups. Compared to the control group, the number of BrdU+ cells of the G-CSF group was significantly increased (p<0.05)
3. With immunofluorescence double-labeled detection tests,Nestin+/BrdU+、MAP-2+/BrdU+ double positive cells could be detected in subventricular zone area, olfactory bulb and hippocampus in both groups. Compared to the control group, the number of Nestin+/BrdU+、MAP-2+/BrdU+ double positive cells in the G-CSF group was significantly increased (p<0.05)
Conclusions:1. G-CSF could induce the migration of HSC into brain.
2. G-CSF could promote the proliferation of cells in the brain of AD mice significantly.
3. G-CSF could induce the differentiation of neural stem cells into neurons in the brain of AD mice.
Significance
This research indicated that subcutaneous injection of G-CSF could improve the cognition in APP transgenic mouse model of Alzheimer's disease. The mechanisms were that G-CSF could mobile the proliferation and differentiation of peripheral HSC and induce the migration into the brain; it also could induce the proliferation and the differentiation of neural stem cells into neurons. In those ways, the loss of neurons could be substituted and the lesions could be repaired. It provided new experimental evidence for the treatment of AD.
阿尔茨海默病(Alzheimer's disease, AD)是一种起病隐袭的中枢神经系统退行性疾病,以记忆力进行性减退、认知功能障碍、行为活动异常为主要临床特征。其患病率约占老年痴呆病例的一半以上,随着人口老龄化的加重,阿尔茨海默病的发病率逐年增高。AD严重影响患者的工作能力和生活质量,也为家庭和社会带来沉重的负担。鉴于AD的发病机制尚未阐明,目前尚无理想的预防和治疗方法。
AD的主要病理特征是在患者大脑皮质、海马、某些皮质下神经核出现老年斑(senile plaque, SP)和神经原纤维缠结(neurofibrillary tangle, NFT),此外还有神经元缺失、星形细胞增生样反应、神经元突触异常等病理改变。AD的核心症状是认知功能进行性下降,谷氨酸能及胆碱能神经元丢失程度与患者智能衰退关系密切。因此,减少神经元丢失或增加神经再生是AD治疗的重要方向。
粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF)是由单核细胞、成纤维细胞和内皮细胞产生的一种造血生长因子,能与细胞表面的特定受体结合,促使中性粒细胞系造血祖细胞生长和分化,保护中性粒细胞避免凋亡并加强它们的功能,G-CSF被广泛地应用于治疗由各种原因引起的粒细胞减少症。最近多项研究显示其对中枢神经系统也有多重效应,可促进骨髓源性干细胞向受损脑区迁移,抗细胞凋亡,促进神经细胞再生。神经干细胞存在大脑特定区域,特别是脑室下区(管膜)、嗅球和海马,这些区域均可表达G-CSF及G-CSFR。G-CSF具有神经保护作用,能促进组织形态修复和神经功能康复。近年来,对成年动物脑缺血模型的研究表明,应用G-CSF是一种有效治疗方法,Kawada等研究发现,缺血性脑卒中动物模型经G-CSF治疗,能通过动员骨髓干细胞而改善卒中后脑功能受损症状。国外应用G-CSF治疗成人缺血性脑卒中已进入Ⅰ、Ⅱ期临床观察阶段。
根据以往研究推测,G-CSF可能通过促进神经再生治疗AD,改善AD患者的认知功能。本研究将G-CSF应用于APPV717I转基因AD模型小鼠,以探讨G-CSF能否促进神经再生和改善转基因小鼠认知能力。本研究共分三部分,如下：
第一部分G-CSF对阿尔茨海默病小鼠认知功能的影响
目的对G-CSF治疗前后AD模型小鼠、对照组和野生小鼠进行生物行为学研究,利用水迷宫模型来测试各组小鼠学习记忆能力,探讨G-CSF能否改善AD小鼠模型的认知能力。
方法1.将10月龄APPV717I转基因AD模型小鼠和野生小鼠分为对照组、G-CSF治疗组和野生组。G-CSF治疗组：连续7d皮下注射G-CSF(50μg/kg·d)。对照组、野生组,连续7d皮下注射PBS。按照G-CSF治疗完成后的天数,各组小鼠又分为三个亚组：7d组,14d组,28d组。
2.各组小鼠于注射前后分别进行水迷宫定位航行试验。
3.利用统计软件对G-CSF治疗前后的AD模型小鼠及野生小鼠的水迷宫实验数据进行统计分析。观察G-CSF对AD模型小鼠认知功能的影响。
结果各组小鼠Morris水迷宫测试结果：AD模型组逃避潜伏期和游泳距离较野生组明显延长(P<0.01),G-CSF治疗组显著缩短潜伏期和游泳距离,和野生组水平相当。同组大鼠,随着训练时段的增加,G-CSF治疗组的潜伏期和游泳距离越来越短(P<0.01)。各组小鼠游泳速度没有明显差异。可以排除因个体差异引起的不同。
结论G-CSF治疗后阿尔茨海默病小鼠认知功能改善。
第二部分G-CSF对阿尔兹海默病小鼠CD 34+/ CD 45+细胞比例的影响
目的应用流式细胞学的方法观测G-CSF治疗前后APP转基因模型小鼠外周血中CD 34+/CD 45+细胞比例,探讨G-CSF对阿尔茨海默病模型小鼠骨髓造血干细胞的影响。
方法1.将10月龄APPV7171转基因AD模型小鼠分为对照组,G-CSF治疗组。G-CSF治疗组：连续7d皮下注射G-CSF(50μg/kg·d)。对照组：连续7d皮下注射PBS。
2.给药第14 d Morris水迷宫测试后从小鼠眶后静脉丛取外周血0.5 ml,进行流式细胞学测试,观察2组小鼠外周血中CD 34+/CD 45+细胞比例。
结果治疗组小鼠外周血CD 34+/CD45+细胞数较对照组明显增加(P<0.01),约为对照组3倍。
结论1.G-CSF能提高AD模型小鼠外周血中CD 34+/CD 45+细胞的比例。
2.G-CSF能够动员AD模型小鼠CD34+的骨髓造血干细胞(hematopoietic stem cells,HSC)增殖并进入外周血。
第三部分G-CSF对阿尔兹海默病小鼠脑内神经元再生的影响
目的对G-CSF治疗前后AD模型小鼠脑组织切片进行免疫组化及免疫荧光双标检测,通过CD 34+免疫荧光染色、Nestin+/BrdU+、MAP-2+/BrdU+免疫荧光双重标记,观察G-CSF能否影响AD小鼠模型脑内的神经再生
方法1G-CSF治疗组：连续7d皮下注射G-CSF(50μg/kg·d)。对照组：连续7d皮下注射PBS。给药同时,两组动物给予BrdU(50 mg/kg·d)腹腔注射,连续10d。
2.在给药后14d将两组小鼠多聚甲醛灌注取脑,冰冻切片,CD 34+免疫荧光染色、Nestin+/BrdU+、MAP-2+/BrdU+免疫荧光双重标记。
结果1.治疗组在给药后第14d时脑内的皮质、嗅球及海马区可检测到红色的CD 34+细胞。
2.在两组小鼠脑内的皮质、嗅球及海马区均检测到BrdU+细胞,治疗组较对照组显著增加(p<0.05)。
3.应用免疫荧光双标染色,在皮质、嗅球及海马区均可见Nestin+/BrdU+、MAP-2+/BrdU+双阳性细胞,较对照组明显增加(p<0.05)。
结论1.G-CSF可诱导AD模型小鼠骨髓造血干细胞向脑内迁移。
2.G-CSF能明显促进AD模型小鼠脑内细胞增殖。
3.G-CSF可诱导AD模型小鼠脑中神经干细胞增殖并向神经元方向分化。
研究意义
本研究结果表明G-CSF皮下注射可明显改善APP转基因小鼠的认知功能,其作用机制为动员外周骨髓造血干细胞增殖、分化,并向脑内定向迁移；同时诱导脑内神经干细胞增殖、向神经元分化,从而替代丢失的神经元,修复病灶,为G-CSF治疗阿尔茨海默病提供了新的实验依据。
Background
Alzheimer's disease (AD) is an insidious-onset neurodegenetative disease of central nervous system (CNS), characterized by progressive memory loss, cognitive dysfunction and behavior disorder. AD occupies more than half of aging dysmnesia. With the increasing proportion of the elderly in the population, its incidence increases year by year. The working ability and life quality of the patients are damaged significantly and this disease places a heavy burden on the family and society. With its pathogenesis still veiled, AD can not be prevented and treated effectively.
The main pathological changes of AD include senile plaque (SP) and neurofibrillary tangle (NFT) in the cerebral cortex, hippocampus and subcortical nucleus, as well as neuron loss, astrocytosis, and the abnormality of neurons and synapsis. The outstanding symptom of AD is progressive cognitive dysfunction, which is closely related to the loss of glutamatergic and cholinergic neurons. Therefore, reducing neuron loss and increasing neuroregeneration is an important trend of AD treatment.
Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor produced by monocyte, fibroblast and endotheliocyte, can bind with specific receptors on the cellular surface, promote the proliferation and differentiation of granulocyte hemopoietic progenitor, protect the neutrophils from apoptosis and intensify their function. G-CSF has been widely used in the treatment of granulocytopenia caused by various reasons. Several recent researches indicated that G-CSF could exert multiple effects on CNS, including inducing marrow-derived stem cells to migrate to the impaired brain area, anti-apoptosis, promoting neurogenesis. Neural stem cells exist in certain areas within the brain, especially subventricular zone area (ependymal), olfactory bulb and hippocampus, where G-CSF and G-CSFR are expressed. G-CSF plays such a neuroprotective role that it promotes the recovery of nervous tissue both functionally and morphologically. Recently, researches on adult animal models of cerebral ischemia showed that G-CSF is an effective drug. Kawada and his team found that the G-CSF treatment for the animal model of ischemic stroke could improve the recovery of the ischemic brain function by mobilizing bone marrow stem cells. The treatment of G-CSF for adult ischemic stroke has entered PhaseⅠandⅡclinical trials abroad.
Based on previous researches, G-CSF might be able to improve cognitive function by promoting neurogenesis. In this research, G-CSF was administrated to APPV7171 transgenic mouse model of AD in order to determine whether G-CSF could induce neuroregeneration and improve the cognition. This research consisted of 3 parts, which would be described as follows.
Part 1. The influence of G-CSF on the cognition of AD mice
Purpose:To study the changes of biological behavior of AD mice after the administration of G-CSF, Morris water maze was used to test their study and memory functions, which was in order to discuss whether G-CSF treatment could improve the cognition of AD mice.
Method:1.Divide 10-month old APPV7171 transgenic AD mice and wild type group randomly into the G-CSF group, the control group and the wild type group. G-CSF group:Subcutaneous injection of G-CSF (50μg/kg·d) for 7 days in succession. Control group and wild type group:Subcutaneous injection of PBS for 7 days in succession. According to the time after the G-CSF treatment, each group was divided into 3 sub-groups:7-day subgroup,14-day subgroup and 28-day subgroup.
2. Each group of mice was under Water maze orientation navigation experiments before and after injection.
3. Analyze the data of Water maze orientation navigation experiments with statistics software. Observe the influence that G-CSF exerts on the AD mice.
Results:Results of Water maze orientation navigation experiments of each group: Comparing to the wild type group, the escape response latency and swimming distance of the AD mice were significantly prolonged; those data of the G-CSF group were shortened significantly and similar with the wild type group. Among mice of the same group, the shortening of the escape response latency and swimming distance increased as the training time extended (P<0.01). There was no significant difference between the swimming speeds of each group. Difference caused by individual variation could be excluded.
Conclusions:The cognitive function of AD mice was improved after the G-CSF treatment.
Part 2 The influence of G-CSF on the CD 34+/CD 45+ratio of AD mice
Purpose:With flow cytometry, we investigated the difference of CD 34+/CD 45+ ratio between the G-CSF group and the control group, which aimed to discuss the influence of G-CSF on the hematopoietic stem cells of AD mice.
Method:1. Divide 10-month old APPV7171 transgenic AD mice randomly into the G-CSF group and the control group. G-CSF group:Subcutaneous injection of G-CSF(50μg/kg·d) for 7 days in succession. Control group:Subcutaneous injection of PBS for 7 days in succession.
2. On the 14th day after the injection, take 0.5ml of peripheral blood from postorbital vein plexus of mice after Morris water maze test and observe the peripheral CD 34+/CD 45+ ration with flow cytometry.
Results:The peripheral CD 34+/CD45+ ratio of G-CSF group was significantly increased compared to the control group (P<0.01), which was about 3 times as large as that of the control group.
Conclusions:1. G-CSF elevated CD 34+/CD 45+ ratio in the peripheral blood of AD mice.
2. G-CSF could promote the proliferation of CD 34+ hematopoietic stem cell (HSC) and mobilize those cells into peripheral blood.
Part 3 The influence of G-CSF on the neurogenesis of AD mice
Purpose:By performing the immunohistochemical and immunofluorescence double-labeled detection tests on the G-CSF group and the control group (immunofluorescence staining of CD 34+, immunofluorescence double-labeling of Nestin+/BrdU+ and MAP-2+/BrdU+), discuss whether G-CSF could influence the neurogenesis.
Method:1. G-CSF group:Subcutaneous injection of G-CSF (50μg/kg·d) for 7 days in succession. Control group:Subcutaneous injection of PBS for 7 days in succession. During the period, both groups were given intraperitoneal injection of BrdU (50 mg/kg·d) for 10 days in succession
2. On the 14th day after the injection, remove the mice brain of both groups by paraformaldehyde perfusion, and frozen on dry ice for the specimen. Then conduct the immunofluorescence staining of CD34+, and immunofluorescence double-labeling of Nestin+/BrdU+, MAP-2+/BrdU+.
Results:1. On the 14th day after the injection, red-stained CD34+ cells were found in subventricular zone area, olfactory bulb and hippocampus of the G-CSF group.
2. BrdU+ cells could be detected in subventricular zone area, olfactory bulb and hippocampus of both groups. Compared to the control group, the number of BrdU+ cells of the G-CSF group was significantly increased (p<0.05)
3. With immunofluorescence double-labeled detection tests,Nestin+/BrdU+、MAP-2+/BrdU+ double positive cells could be detected in subventricular zone area, olfactory bulb and hippocampus in both groups. Compared to the control group, the number of Nestin+/BrdU+、MAP-2+/BrdU+ double positive cells in the G-CSF group was significantly increased (p<0.05)
Conclusions:1. G-CSF could induce the migration of HSC into brain.
2. G-CSF could promote the proliferation of cells in the brain of AD mice significantly.
3. G-CSF could induce the differentiation of neural stem cells into neurons in the brain of AD mice.
Significance
This research indicated that subcutaneous injection of G-CSF could improve the cognition in APP transgenic mouse model of Alzheimer's disease. The mechanisms were that G-CSF could mobile the proliferation and differentiation of peripheral HSC and induce the migration into the brain; it also could induce the proliferation and the differentiation of neural stem cells into neurons. In those ways, the loss of neurons could be substituted and the lesions could be repaired. It provided new experimental evidence for the treatment of AD.
引文
1. Zhao C, Xie Z, Wang P,et al. Granulocyte-colony stimulating factor protects memory impairment in the senescence accelerated mouse (SAM)-P10[J]. Neurol Res,2011,33:354-9.
2.姜昊.粒细胞集落刺激因子治疗阿尔茨海默病抗炎机制的研究.山东大学博士论文.2010,10,25：22.
3. Lippa CF.Clinical characterization in Alzheimer disease[J].Am J Alzheimers Dis Other Demen.2009,24(2):83-4.
4. Fratiglioni L,De Ronchi D,Aguero-Torres H, et al.Worldwide prevalence and incidence of dementia[J].Drugs aging,1999,15(3):65-75.
5. Zhang ZX, Zahner GEP,Roman GC, et al. Dementia Subtypes in China: Prevalence in Beijing, Xian, Shanghai and Chengdu[J]. Arch Neuro,2005,62: 447-53.
6. Wmio A, Winblad B, Jonsson L.An estmiate of the worldwide prevalence and direct costs of dementia in 2005[J].Dement Geriatr Cogn Disord,2006,21(3): 175-81.
7.于大林,肖军.阿尔茨海默病的流行病学调查现况[J].实用医院临床杂志.2011,8(3)：152-5.
8. Standridge JB.Pharmacotherapeutic approaches to the treatment of Alzheimer's disease[J].Clin Ther,2004,26(5):615-30.
9. Daiello LA.Current issues in dementia pharmacotherapy[J].Am J Manag Care.2007,13(Suppl 8):S198-202.
10. Landmark K, Reikvam A.Cholinesterase inhibitors in the treatment of dementia-are they useful in clinical practice[J]? Tidsskr Nor Laegeforen.2008, 128(3):294-7.
11. Hynd MR, Scott HL, Dodd PR.Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease[J].Neurochem Int.2004,45(5):583-95.
12. Herrmann N, Li A, Lanctot K.Memantine in dementia:a review of the current evidence[J].Expert Opin Pharmacother.2011,12(5):787-800.
13. Dantoine T, Auriacombe S, Sarazin M, Becker H,et al.Rivastigmine monotherapy and combination therapy with memantine in patients with moderately severe Alzheimer's disease who failed to benefit from previous cholinesterase inhibitor treatment[J].Int J Clin Pract.2006,60(1):110-8.
14. Maidment ID, Fox CG, Boustani M, Rodriguez J, et al.Efficacy of memantine on behavioral and psychological symptoms related to dementia:a systematic meta-analysis[J]. Ann Pharmacother.2008,42(1):32-8.
15. Rosenberg PB.Clinical aspects of inflammation in Alzheimer's disease[J].Int Rev Psychiatry.2005,17(6):503-14.
16. Birks J, Grimley Evans J. Ginkgo biloba for cognitive impairment and dementia [J].Cochrane Database Syst Rev.2007,18(2):CD003120.
17. Yaffe K, Krueger K, Cummings SR, et al. Effect of raloxifene on prevention of dementia and cognitive impairment in older women:The Multiple Outcomes of Raloxifene Evaluation (MORE) randomized trial [J].Am J Psychiatry,2005, 162(4):683-90.
18. Shumaker SA, Legault C, Rapp SR, et al. Estrogen plus progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women: the Women's Health Initiative Memory Study:a randomized controlled trial [J]. JAMA,2003,289(20):2651-62.
19. Sparks DL, Connor DJ, Sabbagh MN, Petersen RB,et al.Circulating cholesterol levels, apolipoprotein E genotype and dementia severity influence the benefit of atorvastatin treatment in Alzheimer's disease:results of the Alzheimer's Disease Cholesterol-Lowering Treatment (ADCLT) trial[J]. Acta Neurol Scand Suppl. 2006,185:3-7.
20.黄敏,袁进,张相年.阿尔茨海默病临床治疗药物进展[J].临床军医杂 志.2010,38(2)：307-9.
21.徐岚,洪震.阿尔茨海默病的药物治疗进展[J].医药导报.2009,28(9)：1182-4.
22. Rozemuller AJ, van Gool WA, Eikelenboom P. The neuroinflammatory response in plaques and amyloid angiopathy in Alzheimer's disease:therapeutic implications [J]. Curr Drug Targets CNS Neurol Disord.2005,4(3):223-33.
23. Brendza RP, Bacskai BJ, Cirrito JR, Simmons KA.Anti-Abeta antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice[J].J Clin Invest.2005,115 (2):428-33.
24. Aisen PS.The development of anti-amyloid therapy for Alzheimer's disease:from secretase modulators to polymerisation inhibitors [J].CNS Drugs.2005,19(12): 989-96.
25. Duyckaerts C, Delatour B, Potier MC. Classification and basic pathology of Alzheimer disease [J]. Acta Neuropathol,2009,118:5-36.
26.王洁洁,徐家丽.粒细胞集落刺激因子的神经保护作用机制[J].临床儿科杂志.2011,29(4)：389-92.
27. Schneider A,Kruger C,Steigleder T,et al.The hematopoiticfactor G-CSF is a neuronalligand that counteracts programmed cell death and drives neurogesesis [J].Clin Invest,2005,115(8):2083-98.
28. Lu CZ,Xiao BG.G-CSF and neuroprotection:a therapeutic perspective in cerebral ischemia[J].Biochem Soc Trans,2006,34 (Pt 6):1327-33.
29. Kawada H,Takizawa S,Takanashi T,et al.Administration of hematopoietic cytokines in the subacute phase after cerebral infarction is effective for functional recovery facilitating proliferation of intrinsic neural stem/progenitor cells and transition of bone marrow-derived neuronal cells[J].Circulation.2006,113(5):701-10.
30. Yanqing Z,Yu-Min L,Jian Q,et al.Fibronectin and neuroprotective effect of granulocyte colony-stimulating factor in focal cerebral ischemia[J]. Brain Res.2006,1098(1):161-9.
31. Solaroglu I,Tsubokawa T,Cahill J,et al.Anti-apoptotic effect of G-CSF after cerebral ischemia in the rat[J].Neuroscience,2006,143(4):965-74.
32. Yata K,Matchett GA,Tsubokawa T,et al.Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxic-ischemia in rats[J].Brain Res,2007,1145:227-38.
33. Solaroglu I, Cahill J,Tsubokawa T,et al. Granulocyte colonystimulating factor protects the brain against experimental stroke via inhibition of apoptosis and inflammation [J]. Neurol Res,2009,31(2):167-72.
34. Sehara Y,Hayashi T,Deguchi K,et al. Decreased focal inflammatory response by G-CSF may improve stroke outcome after transient middle cerebral artery occlusion in rats [J]. J Neurosci Res,2007,85(10):2167-74.
35. Lee ST,Chu K,Jung KH,et al. Granulocyte colony-stimulating factor enhances angiogenesis after focal cerebral lschemia[J].Brain Res,2005,1058(1-2):120-8.
36. Minamino K,Adachi Y,Okigaki M,et al. Macrophage colonystimulating factor (M-CSF),as well as granulocyte colonystimulating factor (G-CSF), accelerates neovascularization[J].Stem Cells,2005,23(3):347-54.
37. Toth ZE,Leker RR,Shahar T,et al.The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow-derived endothelial cells in brains of mice following cerebral ischemia[J].Blood,2008,111(12):5544-52.
38. DO Wirak,R Bayney,TV Ramabhadran,RP Fracasso,et al.Deposits of amyloid beta protein in the central nervous system of transgenic mice [J].Science(S0036-8075),1991,253(5017):323-5.
39. Games D, Adams D, Alessandrini R, Barbour R,et al.Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein[J].Nature(S0028-0836),1995,373(6514):523-7.
40.常洋,秦川,尹红星,朱华,蔡有余.建立阿尔茨海默症的转基因动物模型[J].解剖学报,31(2000)144-7.
41.黄澜,朱华,高虹,等.不同月龄人App老年痴呆病转基因小鼠行为学观察比较[J].中国老年学杂志,2004,24(12)：1185-6.
42.秦川,朱华,张兵林,等.阿尔茨海默症转基因动物模型脑组织病理学及免疫组化研究[J].中国实验动物学报,2000,8(4)：213-7.
43.张兰,邢颖,赵玲,等.不同时程APP转基因小鼠学习记忆改变及参乌胶囊、二苯乙烯苷的干预作用[J].中国行为医学科学,2006,15(3)：193-6.
44. Morris RGM. Developments of a water-maze procedure for studying spatial learning in the rat [J]. Neurosci Methods,1984,11(1):47-60.
45. Stewart CA, Morris RGM. The water-maze [M]//SahgalA. Behavioural Neuroscience:A Practical Approach. Oxford:IRL Press,1993:107-22.
46. Brandeis R, Brandys R, Yehuda S. The use of the Morris water maze in the study of memory and learning [J]. Neu roscience,1989,48(12):29-69.
47. Vasconcellos AP, Zugno A I, Dos Santos AH. Na+, K(+)-ATPase activity is reduced in hippocampus of rats submitted to an experimental model of depression effect of chronic lithium treatment and possible involvement in learning deficits [J]. Neurobiol Learn Mem,2005,84 (2):102-10.
48. Choeiri C, HewittK, Durk in J. Longitudinal evaluation of memory performance and peripheral neuropathy in the In s2C96Y A kitamice [J]. B ehav Brain Res, 2005,157(1):31-8.
49. R.G. Morris, Episodic-like memory in animals:psychological criteria, neural mechanisms and the value of episodic-like tasks to investigate animal models of neurodegenerative disease[J].Philos Trans R Soc Lond B Biol Sci,356 (2001) 1453-65.
50. Abrahams, A. Pickering, C.E. Polkey, R.G. Morris, Spatial memory deficits in patients with unilateral damage to the right hippocampal formation, Neuropsychologia,35 (1997) 11-24.
51.黎健.干细胞移植治疗心肌梗死[J].中国动脉硬化杂志,2005,13(1)：1-4.
52. Takahashi T, Kalka C, Masuda H, et al. Ischemia and cytokine induced mobilization of bone marrow derived endo the lial progenitor cells for neova scularization [J].NatMed,1999,5:434-8.
53. Shargorodsky M, Leibovitz E, Lubimov L, et al.Prolonged treatment with the AT1 receptor blocker,valsartan, increases small and large artery compliance in uncomplicated essential hypertension[J]..Am J Hypertens,2002,15(12):1087-91.
54. Heissig B, Hatteri K, Dias S, et al.Recruiment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand [J].Cell, 2002,109(5):625-37.
55.杨敏,闫纯英,吴贤仁,等.骨髓干细胞动员后归巢梗死心肌机制研究[J].中国急救医学,2005,25(3)：190-1.
56. Ginis I, Luo Y, Miura T, et al. Diferences between human and mouse embryonic stem cells[J].Dev Biol.2004; 269(2):360-80.
57. Kieusseian A. Are Hematopoietic Stem Cells Generated in the Placenta [J]? Cell Stem Cell.2008; 2(3):197-8.
58.崔若昱.造血干细胞移植治疗下肢缺血性疾病的研究进展[J].河北北方学院学报,2005,22(6)：78-80.
59.任素萍,奚永志.CD34分子及其单克隆抗体应用[J].中国输血杂志,2003,16(5)：350-4.
60.翁建宇,杜欣,张建军,等.G-CSF对供者外周血CD34+细胞黏附分子表达的影响[J].中国输血杂志,2007,20(2)：105-7.
61. yin t,lilh. Thestem cell niche in bone [J].Clin invest,2006,116(3):652-62.
62. Platzbeckeru,ehningerg,bomhauserm,et al. Allogeneictransplantation of cd34+ selected hematopoietic cells and clinical problems and currentchallenges [J].leuk lymphoma,2004,45(3):447-53.
63. Rankin CA, Sun Q, Gamblin TC. Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology. Mol Neurodegener,2007,2:12.
64. Byrne JA,Pedersen DA,Clepper LL,et al.Producing primate embryonic stem cells by somatic cell nuclear transfer[J].Nature,2007,450(7169):497-502.
65. Yamanaka S.Strategies and new developments in the generation of patient-specific pluripotent stem cells[J].Cell Stem Cell,2007,1(1):39-49.
66. Shyu WC, Lin SZ, Chiang MF, et al. Intracerebral peripheral blood stem cell(CD34+) implantation induces neuroplasticity by enhancing (3-integrin-mediated angiogenesis in chronic stroke rats[J].J Neuroscience.2006; 26(13):3444-53.
67. Hennemann B, Ickenstein G, Sauerbruch S,et al. Mobilization of CD34+ hematopoietic cells, colony-forming cells and long-term culture-initiating cells into the peripheral blood of patients with an acute cerebral ischemic insult [J]. Cytotherapy.2008;10(3):303-11.
68.田玉收,李建生,刘敬霞.骨髓干细胞动员抑制脑缺血损伤神经细胞凋亡的研究进展[J].中风与神经疾病杂志,2008,25(2)：250-2.
69.程欣,罗焕敏.干细胞移植治疗阿尔茨海默病的研究进展[J].基础医学与临床,2011,31(2)：218-20.
70.曹云鹏.干细胞治疗阿尔茨海默病的研究进展[J].中国实用内科杂志.2007,27(10)：748-50.
71. Reynolds BA, Weiss S.Generation of neurons and astrocytes from isolated cells of the adultmammalian central nervous system[J].Science,1992,255(5052): 1707-10.
72. Picavd-Riera N,Nait-Oumesmar B, Baron-Van Evercooren A.Endogenous adult neural stem cells:limits and potential to repair the injured central nervous system[J].J Neurosci Res,2004;76(2):223-31.
73. Taupin P. Adult neurogenesis in the mammalian central nervous system: functionality and potential clinical interest [J].Med Sci Monit,2005,11(7):RA 247-52.
74. Haughey NJ,Liu D,Nath A, et al. Disruption of neurogenesis in the subventricular zone of adult mice, and in human cortical neuronal precursor cells in culture, by amyloid beta-peptide implications for the pathogenesis of Alzheimer's disease[J].Neuromolecular Med,2002,1(2):125-35.
75. Sugaya K, Brannen C. Stem cell strategies for neuroreplacement therapy in Alzheimerps disease [J]. Med Hypotheses,2001,57(6):697-700.
76. Sarkis C, Serguera C, Petres S, et al.Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc Natl Acad Sci USA,2000, 97(26):14638-43.
77. Buchet D, Buc-Caron MH, Sabate O, et al. Long-term fate of human telencephalic progenitor cells grafted into the adult mouse brain:effects of previous amplification in vitro [J].J Neurosci Res,2002,68(3):276-83.
78. Jin K, Peel AL,Mao XO,et al.Increased hippocampal neurogenesis in Alzheimer's disease[J].Proc Natl Acad Sci USA,2004; 101(1):343-7.
79. Jin K, GalvanV, Xie L, et al.Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw, Ind) mice [J].Proc Natl Acad Sci USA,2004; 101(36): 13363-7.
80. Ziabreva I, Perry E, Perry R,et al.Altered neurogenesis in Alzheimer's disease[J].J Psychosom Res,2006; 61(3):311-6.
81. Rodriguez JJ, Jones VC, Tabuchi M,et al.Impaired adult neurogenesis in the dentate gyrus of a triple transgenic mouse model of Alzheimer's disease[J].PLoS One,2008; 3(8):e2935.
82. Grote HE,Hannan AJ.Regulators of adult neurogenesis in the healthy and diseased brain[J].Clin Exp Pharmacol Physiol,2007;34(5-6):533-45.
2.姜昊.粒细胞集落刺激因子治疗阿尔茨海默病抗炎机制的研究.山东大学博士论文.2010,10,25：22.
3. Lippa CF.Clinical characterization in Alzheimer disease[J].Am J Alzheimers Dis Other Demen.2009,24(2):83-4.
4. Fratiglioni L,De Ronchi D,Aguero-Torres H, et al.Worldwide prevalence and incidence of dementia[J].Drugs aging,1999,15(3):65-75.
5. Zhang ZX, Zahner GEP,Roman GC, et al. Dementia Subtypes in China: Prevalence in Beijing, Xian, Shanghai and Chengdu[J]. Arch Neuro,2005,62: 447-53.
6. Wmio A, Winblad B, Jonsson L.An estmiate of the worldwide prevalence and direct costs of dementia in 2005[J].Dement Geriatr Cogn Disord,2006,21(3): 175-81.
7.于大林,肖军.阿尔茨海默病的流行病学调查现况[J].实用医院临床杂志.2011,8(3)：152-5.
8. Standridge JB.Pharmacotherapeutic approaches to the treatment of Alzheimer's disease[J].Clin Ther,2004,26(5):615-30.
9. Daiello LA.Current issues in dementia pharmacotherapy[J].Am J Manag Care.2007,13(Suppl 8):S198-202.
10. Landmark K, Reikvam A.Cholinesterase inhibitors in the treatment of dementia-are they useful in clinical practice[J]? Tidsskr Nor Laegeforen.2008, 128(3):294-7.
11. Hynd MR, Scott HL, Dodd PR.Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease[J].Neurochem Int.2004,45(5):583-95.
12. Herrmann N, Li A, Lanctot K.Memantine in dementia:a review of the current evidence[J].Expert Opin Pharmacother.2011,12(5):787-800.
13. Dantoine T, Auriacombe S, Sarazin M, Becker H,et al.Rivastigmine monotherapy and combination therapy with memantine in patients with moderately severe Alzheimer's disease who failed to benefit from previous cholinesterase inhibitor treatment[J].Int J Clin Pract.2006,60(1):110-8.
14. Maidment ID, Fox CG, Boustani M, Rodriguez J, et al.Efficacy of memantine on behavioral and psychological symptoms related to dementia:a systematic meta-analysis[J]. Ann Pharmacother.2008,42(1):32-8.
15. Rosenberg PB.Clinical aspects of inflammation in Alzheimer's disease[J].Int Rev Psychiatry.2005,17(6):503-14.
16. Birks J, Grimley Evans J. Ginkgo biloba for cognitive impairment and dementia [J].Cochrane Database Syst Rev.2007,18(2):CD003120.
17. Yaffe K, Krueger K, Cummings SR, et al. Effect of raloxifene on prevention of dementia and cognitive impairment in older women:The Multiple Outcomes of Raloxifene Evaluation (MORE) randomized trial [J].Am J Psychiatry,2005, 162(4):683-90.
18. Shumaker SA, Legault C, Rapp SR, et al. Estrogen plus progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women: the Women's Health Initiative Memory Study:a randomized controlled trial [J]. JAMA,2003,289(20):2651-62.
19. Sparks DL, Connor DJ, Sabbagh MN, Petersen RB,et al.Circulating cholesterol levels, apolipoprotein E genotype and dementia severity influence the benefit of atorvastatin treatment in Alzheimer's disease:results of the Alzheimer's Disease Cholesterol-Lowering Treatment (ADCLT) trial[J]. Acta Neurol Scand Suppl. 2006,185:3-7.
20.黄敏,袁进,张相年.阿尔茨海默病临床治疗药物进展[J].临床军医杂 志.2010,38(2)：307-9.
21.徐岚,洪震.阿尔茨海默病的药物治疗进展[J].医药导报.2009,28(9)：1182-4.
22. Rozemuller AJ, van Gool WA, Eikelenboom P. The neuroinflammatory response in plaques and amyloid angiopathy in Alzheimer's disease:therapeutic implications [J]. Curr Drug Targets CNS Neurol Disord.2005,4(3):223-33.
23. Brendza RP, Bacskai BJ, Cirrito JR, Simmons KA.Anti-Abeta antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice[J].J Clin Invest.2005,115 (2):428-33.
24. Aisen PS.The development of anti-amyloid therapy for Alzheimer's disease:from secretase modulators to polymerisation inhibitors [J].CNS Drugs.2005,19(12): 989-96.
25. Duyckaerts C, Delatour B, Potier MC. Classification and basic pathology of Alzheimer disease [J]. Acta Neuropathol,2009,118:5-36.
26.王洁洁,徐家丽.粒细胞集落刺激因子的神经保护作用机制[J].临床儿科杂志.2011,29(4)：389-92.
27. Schneider A,Kruger C,Steigleder T,et al.The hematopoiticfactor G-CSF is a neuronalligand that counteracts programmed cell death and drives neurogesesis [J].Clin Invest,2005,115(8):2083-98.
28. Lu CZ,Xiao BG.G-CSF and neuroprotection:a therapeutic perspective in cerebral ischemia[J].Biochem Soc Trans,2006,34 (Pt 6):1327-33.
29. Kawada H,Takizawa S,Takanashi T,et al.Administration of hematopoietic cytokines in the subacute phase after cerebral infarction is effective for functional recovery facilitating proliferation of intrinsic neural stem/progenitor cells and transition of bone marrow-derived neuronal cells[J].Circulation.2006,113(5):701-10.
30. Yanqing Z,Yu-Min L,Jian Q,et al.Fibronectin and neuroprotective effect of granulocyte colony-stimulating factor in focal cerebral ischemia[J]. Brain Res.2006,1098(1):161-9.
31. Solaroglu I,Tsubokawa T,Cahill J,et al.Anti-apoptotic effect of G-CSF after cerebral ischemia in the rat[J].Neuroscience,2006,143(4):965-74.
32. Yata K,Matchett GA,Tsubokawa T,et al.Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxic-ischemia in rats[J].Brain Res,2007,1145:227-38.
33. Solaroglu I, Cahill J,Tsubokawa T,et al. Granulocyte colonystimulating factor protects the brain against experimental stroke via inhibition of apoptosis and inflammation [J]. Neurol Res,2009,31(2):167-72.
34. Sehara Y,Hayashi T,Deguchi K,et al. Decreased focal inflammatory response by G-CSF may improve stroke outcome after transient middle cerebral artery occlusion in rats [J]. J Neurosci Res,2007,85(10):2167-74.
35. Lee ST,Chu K,Jung KH,et al. Granulocyte colony-stimulating factor enhances angiogenesis after focal cerebral lschemia[J].Brain Res,2005,1058(1-2):120-8.
36. Minamino K,Adachi Y,Okigaki M,et al. Macrophage colonystimulating factor (M-CSF),as well as granulocyte colonystimulating factor (G-CSF), accelerates neovascularization[J].Stem Cells,2005,23(3):347-54.
37. Toth ZE,Leker RR,Shahar T,et al.The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow-derived endothelial cells in brains of mice following cerebral ischemia[J].Blood,2008,111(12):5544-52.
38. DO Wirak,R Bayney,TV Ramabhadran,RP Fracasso,et al.Deposits of amyloid beta protein in the central nervous system of transgenic mice [J].Science(S0036-8075),1991,253(5017):323-5.
39. Games D, Adams D, Alessandrini R, Barbour R,et al.Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein[J].Nature(S0028-0836),1995,373(6514):523-7.
40.常洋,秦川,尹红星,朱华,蔡有余.建立阿尔茨海默症的转基因动物模型[J].解剖学报,31(2000)144-7.
41.黄澜,朱华,高虹,等.不同月龄人App老年痴呆病转基因小鼠行为学观察比较[J].中国老年学杂志,2004,24(12)：1185-6.
42.秦川,朱华,张兵林,等.阿尔茨海默症转基因动物模型脑组织病理学及免疫组化研究[J].中国实验动物学报,2000,8(4)：213-7.
43.张兰,邢颖,赵玲,等.不同时程APP转基因小鼠学习记忆改变及参乌胶囊、二苯乙烯苷的干预作用[J].中国行为医学科学,2006,15(3)：193-6.
44. Morris RGM. Developments of a water-maze procedure for studying spatial learning in the rat [J]. Neurosci Methods,1984,11(1):47-60.
45. Stewart CA, Morris RGM. The water-maze [M]//SahgalA. Behavioural Neuroscience:A Practical Approach. Oxford:IRL Press,1993:107-22.
46. Brandeis R, Brandys R, Yehuda S. The use of the Morris water maze in the study of memory and learning [J]. Neu roscience,1989,48(12):29-69.
47. Vasconcellos AP, Zugno A I, Dos Santos AH. Na+, K(+)-ATPase activity is reduced in hippocampus of rats submitted to an experimental model of depression effect of chronic lithium treatment and possible involvement in learning deficits [J]. Neurobiol Learn Mem,2005,84 (2):102-10.
48. Choeiri C, HewittK, Durk in J. Longitudinal evaluation of memory performance and peripheral neuropathy in the In s2C96Y A kitamice [J]. B ehav Brain Res, 2005,157(1):31-8.
49. R.G. Morris, Episodic-like memory in animals:psychological criteria, neural mechanisms and the value of episodic-like tasks to investigate animal models of neurodegenerative disease[J].Philos Trans R Soc Lond B Biol Sci,356 (2001) 1453-65.
50. Abrahams, A. Pickering, C.E. Polkey, R.G. Morris, Spatial memory deficits in patients with unilateral damage to the right hippocampal formation, Neuropsychologia,35 (1997) 11-24.
51.黎健.干细胞移植治疗心肌梗死[J].中国动脉硬化杂志,2005,13(1)：1-4.
52. Takahashi T, Kalka C, Masuda H, et al. Ischemia and cytokine induced mobilization of bone marrow derived endo the lial progenitor cells for neova scularization [J].NatMed,1999,5:434-8.
53. Shargorodsky M, Leibovitz E, Lubimov L, et al.Prolonged treatment with the AT1 receptor blocker,valsartan, increases small and large artery compliance in uncomplicated essential hypertension[J]..Am J Hypertens,2002,15(12):1087-91.
54. Heissig B, Hatteri K, Dias S, et al.Recruiment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand [J].Cell, 2002,109(5):625-37.
55.杨敏,闫纯英,吴贤仁,等.骨髓干细胞动员后归巢梗死心肌机制研究[J].中国急救医学,2005,25(3)：190-1.
56. Ginis I, Luo Y, Miura T, et al. Diferences between human and mouse embryonic stem cells[J].Dev Biol.2004; 269(2):360-80.
57. Kieusseian A. Are Hematopoietic Stem Cells Generated in the Placenta [J]? Cell Stem Cell.2008; 2(3):197-8.
58.崔若昱.造血干细胞移植治疗下肢缺血性疾病的研究进展[J].河北北方学院学报,2005,22(6)：78-80.
59.任素萍,奚永志.CD34分子及其单克隆抗体应用[J].中国输血杂志,2003,16(5)：350-4.
60.翁建宇,杜欣,张建军,等.G-CSF对供者外周血CD34+细胞黏附分子表达的影响[J].中国输血杂志,2007,20(2)：105-7.
61. yin t,lilh. Thestem cell niche in bone [J].Clin invest,2006,116(3):652-62.
62. Platzbeckeru,ehningerg,bomhauserm,et al. Allogeneictransplantation of cd34+ selected hematopoietic cells and clinical problems and currentchallenges [J].leuk lymphoma,2004,45(3):447-53.
63. Rankin CA, Sun Q, Gamblin TC. Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology. Mol Neurodegener,2007,2:12.
64. Byrne JA,Pedersen DA,Clepper LL,et al.Producing primate embryonic stem cells by somatic cell nuclear transfer[J].Nature,2007,450(7169):497-502.
65. Yamanaka S.Strategies and new developments in the generation of patient-specific pluripotent stem cells[J].Cell Stem Cell,2007,1(1):39-49.
66. Shyu WC, Lin SZ, Chiang MF, et al. Intracerebral peripheral blood stem cell(CD34+) implantation induces neuroplasticity by enhancing (3-integrin-mediated angiogenesis in chronic stroke rats[J].J Neuroscience.2006; 26(13):3444-53.
67. Hennemann B, Ickenstein G, Sauerbruch S,et al. Mobilization of CD34+ hematopoietic cells, colony-forming cells and long-term culture-initiating cells into the peripheral blood of patients with an acute cerebral ischemic insult [J]. Cytotherapy.2008;10(3):303-11.
68.田玉收,李建生,刘敬霞.骨髓干细胞动员抑制脑缺血损伤神经细胞凋亡的研究进展[J].中风与神经疾病杂志,2008,25(2)：250-2.
69.程欣,罗焕敏.干细胞移植治疗阿尔茨海默病的研究进展[J].基础医学与临床,2011,31(2)：218-20.
70.曹云鹏.干细胞治疗阿尔茨海默病的研究进展[J].中国实用内科杂志.2007,27(10)：748-50.
71. Reynolds BA, Weiss S.Generation of neurons and astrocytes from isolated cells of the adultmammalian central nervous system[J].Science,1992,255(5052): 1707-10.
72. Picavd-Riera N,Nait-Oumesmar B, Baron-Van Evercooren A.Endogenous adult neural stem cells:limits and potential to repair the injured central nervous system[J].J Neurosci Res,2004;76(2):223-31.
73. Taupin P. Adult neurogenesis in the mammalian central nervous system: functionality and potential clinical interest [J].Med Sci Monit,2005,11(7):RA 247-52.
74. Haughey NJ,Liu D,Nath A, et al. Disruption of neurogenesis in the subventricular zone of adult mice, and in human cortical neuronal precursor cells in culture, by amyloid beta-peptide implications for the pathogenesis of Alzheimer's disease[J].Neuromolecular Med,2002,1(2):125-35.
75. Sugaya K, Brannen C. Stem cell strategies for neuroreplacement therapy in Alzheimerps disease [J]. Med Hypotheses,2001,57(6):697-700.
76. Sarkis C, Serguera C, Petres S, et al.Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc Natl Acad Sci USA,2000, 97(26):14638-43.
77. Buchet D, Buc-Caron MH, Sabate O, et al. Long-term fate of human telencephalic progenitor cells grafted into the adult mouse brain:effects of previous amplification in vitro [J].J Neurosci Res,2002,68(3):276-83.
78. Jin K, Peel AL,Mao XO,et al.Increased hippocampal neurogenesis in Alzheimer's disease[J].Proc Natl Acad Sci USA,2004; 101(1):343-7.
79. Jin K, GalvanV, Xie L, et al.Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw, Ind) mice [J].Proc Natl Acad Sci USA,2004; 101(36): 13363-7.
80. Ziabreva I, Perry E, Perry R,et al.Altered neurogenesis in Alzheimer's disease[J].J Psychosom Res,2006; 61(3):311-6.
81. Rodriguez JJ, Jones VC, Tabuchi M,et al.Impaired adult neurogenesis in the dentate gyrus of a triple transgenic mouse model of Alzheimer's disease[J].PLoS One,2008; 3(8):e2935.
82. Grote HE,Hannan AJ.Regulators of adult neurogenesis in the healthy and diseased brain[J].Clin Exp Pharmacol Physiol,2007;34(5-6):533-45.