粒细胞集落刺激因子治疗阿尔茨海默病抗炎机制的研究
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摘要
阿尔茨海默氏症(Alzheimer's disease, AD)又称老年痴呆症,是以德国精神病学家Alois Alzheimer名字命名的一种老年神经变性综合症。该病以记忆逐步减退和进行性认知障碍为临床特征。年龄是AD的重要危险因素。由于人均寿命延长,老年人口迅速增长,AD患病人数必然相应增加。我国65岁以上老人中AD患病率为0.3‰左右,已与发达国家接近。AD自1906年发现至今已有百多年历史,迄今仍未有有效治疗方法。该病不仅造成老年人群生活质量下降,同时也给社会和家庭带来沉重的经济和心理负担,成为老龄社会中主要卫生保健和社会问题。所以寻找有切实有效的治疗药物一直是AD研究领域的重要课题。
据目前细胞学和分子生物学的研究普遍认为,以β-淀粉样蛋白(p-amyloid protein, Aβ)为主要成分的神经元缠结(neurofibrillary tangles, NFTs)和老年斑(senile plaques, SP)形成是引起脑内胆碱能神经元的功能障碍和死亡的主要原因。Aβ与小胶质细胞表面的清道夫受体或糖基化终产物受体结合,激活小胶质细胞,导致炎性因子的释放。这些炎性因子如：IL-1、TNF-αIL-6等造成神经元的功能和行为障碍。最近的证据表明,炎性介质会通过上调β-分泌酶刺激淀粉样前体蛋白(Amyloid precursor protein, APP)的加工过程并形成一个恶性循环,使病情不断进展。有多篇文献阐明,非甾体类抗炎药可以降低患AD的危险性并延缓发病,由此可以推断：慢性炎症过程是AD病理过程中的重要机制,抗炎是治疗AD的切实可行的措施。
粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF),是由174个氨基酸组成的多肽和4%葡萄糖苷型糖链组成的糖蛋白。主要在中性粒细胞及神经细胞和胶质细胞表达。它的主要作用有：1促增殖与分化,2抗炎,3抗凋亡。最近的研究证实,G-CSF具有神经保护的作用,作用机理主要是通过减少致炎因子的释放,而发挥神经保护作用。TNF和IL-1p是炎症中的主要调节因子,G-CSF主要通过对两者的抑制作用发挥它的抗炎作用,其机制不明。
烟碱型胆碱能受体(Nicotinic acetylcholine receptors, nAChRs)是配体门控性离子通道,由五个亚单位构成。α7nAChR是脊椎动物脑中分布最广的两类nAchR亚型之一,在神经突触传递过程中其重要作用。近年来研究发现,a7nAChR表达增高能够降低致炎因子的分泌,是胆碱能抗炎通路的关键受体。a7nAChR的表达增高能够改善AD患者的认知能力。
根据已往的研究发现,G-CSF可能通过抑制炎症治疗AD。作为最新的治疗AD的策略,本研究将G-CSF应用于APPV717I转基因AD模型小鼠,以探讨G-CSF的抗炎作用和可能的抗炎机制,为今后对AD的治疗提供理论依据和事实依据。本研究共分四部分进行。
第一部分G-CSF对阿尔茨海默病小鼠的认知功能的影响
目的对G-CSF治疗前后老年痴呆症模型小鼠和野生小鼠进行生物行为学研究,通过水迷宫测试对老年痴呆症模型小鼠和野生小鼠的学习记忆功能进行观察,探讨G-CSF对老年痴呆症模型小鼠和野生小鼠的认知功能的影响。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belancedsolution；磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.各组小鼠于注射前后分别进行水迷宫定位航行试验。
3.取个时间点小鼠脑组织制作病理切片,用HE (haematoxylin&eosin)和Nissl染色进行形态学观察。
4.利用统计软件对G-CSF治疗前后的老年痴呆模型小鼠和野生型老龄小鼠的水迷宫实验数据进行统计分析。观察G-CSF对老年痴呆模型小鼠的认知的影响。
结果
1.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。
2.HE及Nissl染色示野生小鼠海马神经元结构清晰完整,细胞核结构正常,染色质分布均匀,胞浆内尼氏小体丰富。AD模型组可观察到小鼠海马神经元损伤,表现为神经元肿胀、破裂,排列紊乱,胞浆内尼氏小体减少,存活神经元数目较对照组减少(P<0.05)。G-CSF治疗组海马神经元结构与野生组相似。
结论
1.阿尔茨海默病小鼠认知功能减退。
2. G-CSF治疗后阿尔茨海默病小鼠认知功能改善。
3. G-CSF治疗对阿尔茨海默病小鼠海马神经元有保护作用。
第二部分G-CSF对阿尔茨海默病小鼠脑内炎症的影响
目的研究G-CSF对APPV717I转基因阿尔茨海默病小鼠脑内炎症的影响。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belanced solution;磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.小鼠分别在各时间点灌注多聚甲醛后取脑,免疫荧光的方法检测小鼠大脑皮层细胞的IL-1β. TNF-a表达变化及G-CSF对其表达的影响。
结果
1.图像分析结果显示,AD模型7天组、AD模型14天组、AD模型28天组的IL-1β光密度值均高于同一天的G-CSF治疗组和野生组,且有统计学意义(P<0.01或0.05)；三组比较有显著性差异(P<0.05)。
2.图像分析结果显示,AD模型14天组、AD模型28天组的TNF-a光密度值均高于同一天的G-CSF治疗组和野生组,且有统计学意义(P<0.01或0.05)；三组比较有显著性差异(P<0.05)。
结论
1.阿尔茨海默病模型小鼠脑内炎症因子IL-1β和TNF-a含量相对于野生型同龄小鼠有明显升高。
2. G-CSF治疗,可以降低阿尔茨海默病模型小鼠脑内炎症因子IL-1β和TNF-a含量。
3.推测阿尔茨海默病模型小鼠脑内可能伴随炎症反应,这一反应参与了老年痴呆模型小鼠脑内神经元的损伤过程。而G-CSF彳很可能通过抑制阿尔茨海默病模型小鼠的脑内炎性因子IL-1β、TNF-a的表达而抑制脑内的神经炎症反应,对阿尔茨海默病模型小鼠的大脑皮层神经元起一定的保护作用。
第三部分G-CSF对阿尔茨海默病小鼠脑内的乙酰胆碱受体α7表达的影响
目的研究G-CSF对阿尔茨海默病小鼠脑内的a7nAChR的表达水平的影响,探讨a7nAChR在胆碱能通路中的关键地位。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belancedsolution;磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.各组小鼠分别在各时间点灌注多聚甲醛后取脑,制作冰冻切片。用免疫组化染色的方法检测小鼠大脑皮层细胞α7nAChR的蛋白表达水平变化及G-CSF对其表达的影响。
2.将各组小鼠分别在各时间点断头取脑,脑组织-80℃存储待用。
3.应用Western blotting方法检测各亚组小鼠大脑α7nAChR的蛋白表达水平。
结果
(1)图像分析结果显示,G-CSF治疗7天组、G-CSF治疗14天组、G-CSF治疗28天组的α7nAChR光密度值均高于同一天的AD模型组和野生组,且有统计学意义(P<0.01或0.05)；三组比较有显著性差异(P<0.05)。
(2)α7nAChR蛋白表达比较：G-CSF治疗7天组、G-CSF治疗14天组、G-CSF治疗28天组的α7nAChR蛋白表达均高于同一天的AD模型组和野生组,且有统计学意义(P<0.01),同一天AD模型组α7nAChR蛋白的表达较野生组明显降低(P<0.01)；三组比较有显著性差异(P<0.05)。
结论
1.α7nAChR在各亚组小鼠大脑皮层细胞普遍表达。
2.阿尔茨海默病小鼠大脑皮层细胞α7nAChR的蛋白表达水平明显降低。
3.应用G-CSF能够明显增加各亚组小鼠大脑皮层细胞α7nAChR的蛋白表达水平,对阿尔茨海默病小鼠具有神经保护功能。
第四部分G-CSF对NF-kB的表达的影响及炎症机制的研究
目的研究G-CSF对阿尔茨海默病小鼠脑内的NF-kB的表达水平的影响以及NF-kB表达水平与α7nAChR表达的关系,探讨α7nAChR在胆碱能通路中的关键地位。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belanced solution;磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.小鼠分别在各时间点灌注多聚甲醛后取脑,免疫荧光的方法检测小鼠大脑皮层细胞的NF-kB表达变化及G-CSF对其表达的影响。
3.用统计学的方法对小鼠大脑皮层细胞的NF-kB表达变化和a7nAChR表达变化进行相关性分析。
结果
相关性分析显示G-CSF治疗后AD模型小鼠7天组、14天组、28天组脑内NF-kB和α7nAChR表达成负相关(7天组：r=-0.73,p=0.007；14天组：r=-0.83,p=0.008；28天组：r=-0.89,p=0.02,)。
结论
G-CSF治疗后AD模型小鼠脑内NF-kB的表达水平与α7nAChR的表达水平成负相关,说明G-CSF治疗的抗炎机制是通过增加a7nAChR的表达,降低了NF-kB表达,从而减少致炎因子的转录。
Background
Alzheimer'S disease named as a German psychiatrist, is a neurodegenerative disorders correlated wim age, approximately 60 percent in dementia. More and more olds are bothered by AD, and AD has been the fourth cause of old people's death next to cardiovascular diseases、cancer、stroke. This disease not only cause the decline of living levels of old people but also bring the burden of enconomy and mentality to the society and family. So, it is most improtant for us to find the effective treatment of AD.
Accroding to the current research of cytology and microbiology,β-amyloid protein is the main reason of the death of neurons.β-amyloid protein is composed of neurofibrillary tangles and senile plaques. Aβcombined with receptors on microglia can active the microgalia and cause the secrete of pro-inflammatory factors, such as, IL-1, TNF-α, IL-6. They will cause the function and behavioral disturbance. Latest proofs indicated that mediators of inflammation will aggravate the inflammation processing through up-regulate theβ-secretase.Many reports indicated that anti-inflammatory agents may degrade the risk of suffer AD. To conclusion, chronic inflammation is the main pathology of AD. Anti-inflammation should be the potential treatment of AD.
Granulocyte colony-stimulating factor (G-CSF), a hematopoietic factor, is a stimulator of neutrophil differentiation, proliferation and function. It is used clinically to treat neutropenia. Recently, reports described G-CSF with anti-apoptosis, anti-inflammation, neurotrophy, and angiogenesis roles related to neuroprotection. G-CSF decreases the levels of interleukin-1β(IL-1β), IL-6, and IL-8 in several conditions. G-CSF administration can rescue cognitive abilities through anti-inflammation in AD and in mice models of cerebral ischemia, but the precise mechanism of G-CSF reducing the secretion of proinflammatory cytokines remain unknown.
Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels widely expressed in mammalian cells. They are composed of a diverse pentameric assembly ofα-andβ-subunits. The subtype a7 nAChR is one of the predominant nAChRs in the brain. It is extensively expressed in microglia, astrocytes and neurons.α7 nAChR plays a crucial role in modulating postsynaptic transmission. Moreover,α7 nAChR decreases the level of inflammatory mediators, including TNF-a, IL-1β, IL-6, and IL-18. As a regulator of neuroinflammation, the increased expression of a7 nAChR can attenuate the expression of pro-inflammation. Up-regulate the expression ofα7 nAChR could improve the cognitive ability of AD patients.
G-CSF can cure AD via anti-inflammatory. As the new strategy of AD treatment, we will investigate its ability of anti-inflammatory and possiable mechanisms of anti-inflammation. These will provide evidents for the cure of AD. There are four parts of my research:
Part 1 Influence of G-CSF on the cognitive function of the Alzheimer's disease mice model
Objective:To investigate ethology and pathological changes of Alzheimers disease mice model and wild mice, Morris water maze was used to evaluate their abilities of spatial learning and memory. This research discussed the influence of the G-CSF upon the cognition function of Alzheimers disease mice model and wild mice.
Methods:
1. Alzheimers disease mice model and wild mice were divided into G-CSF group, control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. The mice were given Morris water maze examination before and after G-CSF administration.
3. Mice were perfused with 4% paraformaldehyde and the brains were used to made pathological sections. Hippocampal pathological changes were observed with haematoxylin & eosin (HE) and Nissl staining.
4. To observe the influence of the G-CSF upon the cognition function of Alzheimers disease mice model and wild mice, statistics software was used to analyse the experimental statements of Morris water maze examination before and after G-CSF administration.
Results
1.Morris water maze:Spatial acquisition:In the spatial acquisition phase Of testing, Control group had significantly longer swim time and swim distant than wild type group(P<0.01). Compare to Control group, G-CSF group sigficantly reduced swim time and swim distant, which was close to swim time and swim distant of wild type group. Moreover, there Was a significantly decreased trend for swim time and swim distant in G-CSF group(P＜0.01). There Was no effect of block or treatment on swim speed, and no treatment by block interaction. In the Spatial retention trial, both quadrant time and platform crossings,There was no statistical difference between wild type group and G-CSF group(P<0.05) (Figurel-4).
2. HE and Nissl staining show a clear structure of hippocampus neurons.The cell nucleus structure is normal.The Nissl's bodies in the intracytoplasm were abundant.The chromatins of cell nucleus were distributed equally. Hippocampus neurons of Alzheimer's disease mice model were injuried.Swelled neurons and broken neurons were observed and they ranked chaos in the sections.The Nissl's bodies in the intracytoplasm and survival neurons were reduced compared with control groups (P<0.05).The structure of hippocampus neurons were similar as the wild type groups (Figure5,6,Tablel,2)
Conclusions
1. The cognition function of Alzheimer's disease mice model was decreased.
2. The cognition function of Alzheimer's disease mice model was improved after the administration of G-CSF.
3. G-CSF administration was benefited for the cognitive function of Alzheimer's disease mice model.
Part 2 Influence of G-CSF on the neuroinflammation in the brain of Alzheimer's disease mice model
Objective
Investigate the influence of G-CSF on the neuroinflammation in the brain of Alzheimer's disease mice model.
Methods:
1. Alzheimers disease mice model and wild mice were divided into G-CSF group、control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of IL-1βand TNF-a in mice's frontal lobe cortex was detected by immunofluorescence, and observe the impact of G-CSF on their expression.
Results
1. Analysis result of image showed that the relative immunofluorescence intensity levels of IL-1βin the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P＜0.05) (Figurel,2). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figurel,.2). There is significate difference bwteen three groups (P<0.05).
2. Analysis result of image showed that the relative immunofluorescence intensity levels of TNF-a in the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P<0.05) (Figure3,4). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figure3,4). There is significate difference bwteen three groups (P<0.05).
Conclusions
1. It was confirmed in my research that neuroinflammation exist in the brain of Alzheimer's mice model.
2. MDL-28170 protects hippocampal neuron after pilocarpine-induced seizure against PCD through the inhibited effect onμ-calpain.
3. The administration of G-CSF attenuate neuroinflammtion in Alzheimer's mice model through repress the expression of inflammatory factors IL-1βand TNF-a. It protected the cortical neurons from neuroinflammation of Alzheimer's disease mice model.
Part 3 Influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model
Objective
Investigate the influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model. Discuss the critical effect of a7 nAChR on the cholinergic anti-inflammatory pathway.
Methods:
1. Alzheimers disease mice model and wild mice were divided into G-CSF group、control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of a7 nAChR in mice's frontal lobe cortex was detected by immunohistochemistry (IHC), and observed the impact of G-CSF on their expression.
3. Mice were sacrificed at different time-points. Their brains were obtained and stored at-80℃.
4. The proteins expression of a7 nAChR was detected by Western blotting and the effects of G-CSF on their expression were observed.
Results
1.Analysis result of image showed that density values ofα7nAChR in G-CSF day 7 group, G-CSF day 14 group, G-CSF day 28 group, G-CSF day 56 group were higher statistically than that of control group and wild type group(P<0.01 or 0.05). There is significant difference between the three groups (P<0.05) (Figurel,2).
2.Comparison of protein levels ofα7nAChR:The protein levels of a7nAChR protein in G-CSF group increased significantly compared to that in control group(P<0.01).There is significant difference between the three groups (P<0.05) (Figure3,4).
Conclusion
1.α7 nAChR expressed generally on the front lobe cortex of mice's brain.
2. The expression of a7 nAChR on the front lobe cortex of Alzheimer's mice model's brain was significately depressed.
3. The administration of G-CSF could increase the expression of a7 nAChR on the brain of Alzheimer's mice model in different time points. It has the function of neuroprotection.
Part 4 Influence of G-CSF on the expression NF-kB in the brain of Alzheimer's disease mice model and study of inflammation mechanisms
Objective
Investigate the influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model. Discuss the critical effect of a7 nAChR on the cholinergic anti-inflammatory pathway.
Methods
1. Alzheimers disease mice model and wild mice were divided into G-CSF group,control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of NF-κB in mice's frontal lobe cortex was detected by immunofluorescence, and observed the impact of G-CSF on their expression.
3. Spearman correlation analysis was used to analysis the relationship of the expression ofα7 nAChR and NF-κB at different time points.
Results
1. Analysis result of image showed that the relative immunofluorescence intensity levels of NF-κB in the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P<0.05) (Figurel,.2). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figurel,.2). There is significate difference bwteen three groups (P<0.05).
2. Result of Spearman correlations analysis showed that there was significant negative correlations betweenα7 nAChR and NF-κB expressions (7d:rho=-0.73, p=0.007; 14d:rho=-0.83, p=0.008; 28d:rho=-0.89, p=0.02).
Conclusion there is an inverse correlation between the expression ofα7 nAChR and NF-κB in the brain of Alzheimer's disease mice model after the treatment of G-CSF. This finding suggests that G-CSF significantly attenuates the expression of proinflammatory cytokines by increasing the expression ofα7 nAChR in the brain of APP transgenic mice.
据目前细胞学和分子生物学的研究普遍认为,以β-淀粉样蛋白(p-amyloid protein, Aβ)为主要成分的神经元缠结(neurofibrillary tangles, NFTs)和老年斑(senile plaques, SP)形成是引起脑内胆碱能神经元的功能障碍和死亡的主要原因。Aβ与小胶质细胞表面的清道夫受体或糖基化终产物受体结合,激活小胶质细胞,导致炎性因子的释放。这些炎性因子如：IL-1、TNF-αIL-6等造成神经元的功能和行为障碍。最近的证据表明,炎性介质会通过上调β-分泌酶刺激淀粉样前体蛋白(Amyloid precursor protein, APP)的加工过程并形成一个恶性循环,使病情不断进展。有多篇文献阐明,非甾体类抗炎药可以降低患AD的危险性并延缓发病,由此可以推断：慢性炎症过程是AD病理过程中的重要机制,抗炎是治疗AD的切实可行的措施。
粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF),是由174个氨基酸组成的多肽和4%葡萄糖苷型糖链组成的糖蛋白。主要在中性粒细胞及神经细胞和胶质细胞表达。它的主要作用有：1促增殖与分化,2抗炎,3抗凋亡。最近的研究证实,G-CSF具有神经保护的作用,作用机理主要是通过减少致炎因子的释放,而发挥神经保护作用。TNF和IL-1p是炎症中的主要调节因子,G-CSF主要通过对两者的抑制作用发挥它的抗炎作用,其机制不明。
烟碱型胆碱能受体(Nicotinic acetylcholine receptors, nAChRs)是配体门控性离子通道,由五个亚单位构成。α7nAChR是脊椎动物脑中分布最广的两类nAchR亚型之一,在神经突触传递过程中其重要作用。近年来研究发现,a7nAChR表达增高能够降低致炎因子的分泌,是胆碱能抗炎通路的关键受体。a7nAChR的表达增高能够改善AD患者的认知能力。
根据已往的研究发现,G-CSF可能通过抑制炎症治疗AD。作为最新的治疗AD的策略,本研究将G-CSF应用于APPV717I转基因AD模型小鼠,以探讨G-CSF的抗炎作用和可能的抗炎机制,为今后对AD的治疗提供理论依据和事实依据。本研究共分四部分进行。
第一部分G-CSF对阿尔茨海默病小鼠的认知功能的影响
目的对G-CSF治疗前后老年痴呆症模型小鼠和野生小鼠进行生物行为学研究,通过水迷宫测试对老年痴呆症模型小鼠和野生小鼠的学习记忆功能进行观察,探讨G-CSF对老年痴呆症模型小鼠和野生小鼠的认知功能的影响。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belancedsolution；磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.各组小鼠于注射前后分别进行水迷宫定位航行试验。
3.取个时间点小鼠脑组织制作病理切片,用HE (haematoxylin&eosin)和Nissl染色进行形态学观察。
4.利用统计软件对G-CSF治疗前后的老年痴呆模型小鼠和野生型老龄小鼠的水迷宫实验数据进行统计分析。观察G-CSF对老年痴呆模型小鼠的认知的影响。
结果
1.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。
2.HE及Nissl染色示野生小鼠海马神经元结构清晰完整,细胞核结构正常,染色质分布均匀,胞浆内尼氏小体丰富。AD模型组可观察到小鼠海马神经元损伤,表现为神经元肿胀、破裂,排列紊乱,胞浆内尼氏小体减少,存活神经元数目较对照组减少(P<0.05)。G-CSF治疗组海马神经元结构与野生组相似。
结论
1.阿尔茨海默病小鼠认知功能减退。
2. G-CSF治疗后阿尔茨海默病小鼠认知功能改善。
3. G-CSF治疗对阿尔茨海默病小鼠海马神经元有保护作用。
第二部分G-CSF对阿尔茨海默病小鼠脑内炎症的影响
目的研究G-CSF对APPV717I转基因阿尔茨海默病小鼠脑内炎症的影响。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belanced solution;磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.小鼠分别在各时间点灌注多聚甲醛后取脑,免疫荧光的方法检测小鼠大脑皮层细胞的IL-1β. TNF-a表达变化及G-CSF对其表达的影响。
结果
1.图像分析结果显示,AD模型7天组、AD模型14天组、AD模型28天组的IL-1β光密度值均高于同一天的G-CSF治疗组和野生组,且有统计学意义(P<0.01或0.05)；三组比较有显著性差异(P<0.05)。
2.图像分析结果显示,AD模型14天组、AD模型28天组的TNF-a光密度值均高于同一天的G-CSF治疗组和野生组,且有统计学意义(P<0.01或0.05)；三组比较有显著性差异(P<0.05)。
结论
1.阿尔茨海默病模型小鼠脑内炎症因子IL-1β和TNF-a含量相对于野生型同龄小鼠有明显升高。
2. G-CSF治疗,可以降低阿尔茨海默病模型小鼠脑内炎症因子IL-1β和TNF-a含量。
3.推测阿尔茨海默病模型小鼠脑内可能伴随炎症反应,这一反应参与了老年痴呆模型小鼠脑内神经元的损伤过程。而G-CSF彳很可能通过抑制阿尔茨海默病模型小鼠的脑内炎性因子IL-1β、TNF-a的表达而抑制脑内的神经炎症反应,对阿尔茨海默病模型小鼠的大脑皮层神经元起一定的保护作用。
第三部分G-CSF对阿尔茨海默病小鼠脑内的乙酰胆碱受体α7表达的影响
目的研究G-CSF对阿尔茨海默病小鼠脑内的a7nAChR的表达水平的影响,探讨a7nAChR在胆碱能通路中的关键地位。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belancedsolution;磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.各组小鼠分别在各时间点灌注多聚甲醛后取脑,制作冰冻切片。用免疫组化染色的方法检测小鼠大脑皮层细胞α7nAChR的蛋白表达水平变化及G-CSF对其表达的影响。
2.将各组小鼠分别在各时间点断头取脑,脑组织-80℃存储待用。
3.应用Western blotting方法检测各亚组小鼠大脑α7nAChR的蛋白表达水平。
结果
(1)图像分析结果显示,G-CSF治疗7天组、G-CSF治疗14天组、G-CSF治疗28天组的α7nAChR光密度值均高于同一天的AD模型组和野生组,且有统计学意义(P<0.01或0.05)；三组比较有显著性差异(P<0.05)。
(2)α7nAChR蛋白表达比较：G-CSF治疗7天组、G-CSF治疗14天组、G-CSF治疗28天组的α7nAChR蛋白表达均高于同一天的AD模型组和野生组,且有统计学意义(P<0.01),同一天AD模型组α7nAChR蛋白的表达较野生组明显降低(P<0.01)；三组比较有显著性差异(P<0.05)。
结论
1.α7nAChR在各亚组小鼠大脑皮层细胞普遍表达。
2.阿尔茨海默病小鼠大脑皮层细胞α7nAChR的蛋白表达水平明显降低。
3.应用G-CSF能够明显增加各亚组小鼠大脑皮层细胞α7nAChR的蛋白表达水平,对阿尔茨海默病小鼠具有神经保护功能。
第四部分G-CSF对NF-kB的表达的影响及炎症机制的研究
目的研究G-CSF对阿尔茨海默病小鼠脑内的NF-kB的表达水平的影响以及NF-kB表达水平与α7nAChR表达的关系,探讨α7nAChR在胆碱能通路中的关键地位。
方法
1.10月龄APP V717转基因老年痴呆模型小鼠和10月龄野生小鼠,分为对照组、G-CSF治疗组和野生组。对照组连续7天皮下注射PBS (phosphate belanced solution;磷酸盐缓冲液)；G-CSF治疗组连续7天皮下注射G-CSF;野生组连续7天皮下注射PBS。按照G-CSF治疗完成后的天数,三组小鼠又分为三个亚组：7天组、14天组、28天组。
2.小鼠分别在各时间点灌注多聚甲醛后取脑,免疫荧光的方法检测小鼠大脑皮层细胞的NF-kB表达变化及G-CSF对其表达的影响。
3.用统计学的方法对小鼠大脑皮层细胞的NF-kB表达变化和a7nAChR表达变化进行相关性分析。
结果
相关性分析显示G-CSF治疗后AD模型小鼠7天组、14天组、28天组脑内NF-kB和α7nAChR表达成负相关(7天组：r=-0.73,p=0.007；14天组：r=-0.83,p=0.008；28天组：r=-0.89,p=0.02,)。
结论
G-CSF治疗后AD模型小鼠脑内NF-kB的表达水平与α7nAChR的表达水平成负相关,说明G-CSF治疗的抗炎机制是通过增加a7nAChR的表达,降低了NF-kB表达,从而减少致炎因子的转录。
Background
Alzheimer'S disease named as a German psychiatrist, is a neurodegenerative disorders correlated wim age, approximately 60 percent in dementia. More and more olds are bothered by AD, and AD has been the fourth cause of old people's death next to cardiovascular diseases、cancer、stroke. This disease not only cause the decline of living levels of old people but also bring the burden of enconomy and mentality to the society and family. So, it is most improtant for us to find the effective treatment of AD.
Accroding to the current research of cytology and microbiology,β-amyloid protein is the main reason of the death of neurons.β-amyloid protein is composed of neurofibrillary tangles and senile plaques. Aβcombined with receptors on microglia can active the microgalia and cause the secrete of pro-inflammatory factors, such as, IL-1, TNF-α, IL-6. They will cause the function and behavioral disturbance. Latest proofs indicated that mediators of inflammation will aggravate the inflammation processing through up-regulate theβ-secretase.Many reports indicated that anti-inflammatory agents may degrade the risk of suffer AD. To conclusion, chronic inflammation is the main pathology of AD. Anti-inflammation should be the potential treatment of AD.
Granulocyte colony-stimulating factor (G-CSF), a hematopoietic factor, is a stimulator of neutrophil differentiation, proliferation and function. It is used clinically to treat neutropenia. Recently, reports described G-CSF with anti-apoptosis, anti-inflammation, neurotrophy, and angiogenesis roles related to neuroprotection. G-CSF decreases the levels of interleukin-1β(IL-1β), IL-6, and IL-8 in several conditions. G-CSF administration can rescue cognitive abilities through anti-inflammation in AD and in mice models of cerebral ischemia, but the precise mechanism of G-CSF reducing the secretion of proinflammatory cytokines remain unknown.
Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels widely expressed in mammalian cells. They are composed of a diverse pentameric assembly ofα-andβ-subunits. The subtype a7 nAChR is one of the predominant nAChRs in the brain. It is extensively expressed in microglia, astrocytes and neurons.α7 nAChR plays a crucial role in modulating postsynaptic transmission. Moreover,α7 nAChR decreases the level of inflammatory mediators, including TNF-a, IL-1β, IL-6, and IL-18. As a regulator of neuroinflammation, the increased expression of a7 nAChR can attenuate the expression of pro-inflammation. Up-regulate the expression ofα7 nAChR could improve the cognitive ability of AD patients.
G-CSF can cure AD via anti-inflammatory. As the new strategy of AD treatment, we will investigate its ability of anti-inflammatory and possiable mechanisms of anti-inflammation. These will provide evidents for the cure of AD. There are four parts of my research:
Part 1 Influence of G-CSF on the cognitive function of the Alzheimer's disease mice model
Objective:To investigate ethology and pathological changes of Alzheimers disease mice model and wild mice, Morris water maze was used to evaluate their abilities of spatial learning and memory. This research discussed the influence of the G-CSF upon the cognition function of Alzheimers disease mice model and wild mice.
Methods:
1. Alzheimers disease mice model and wild mice were divided into G-CSF group, control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. The mice were given Morris water maze examination before and after G-CSF administration.
3. Mice were perfused with 4% paraformaldehyde and the brains were used to made pathological sections. Hippocampal pathological changes were observed with haematoxylin & eosin (HE) and Nissl staining.
4. To observe the influence of the G-CSF upon the cognition function of Alzheimers disease mice model and wild mice, statistics software was used to analyse the experimental statements of Morris water maze examination before and after G-CSF administration.
Results
1.Morris water maze:Spatial acquisition:In the spatial acquisition phase Of testing, Control group had significantly longer swim time and swim distant than wild type group(P<0.01). Compare to Control group, G-CSF group sigficantly reduced swim time and swim distant, which was close to swim time and swim distant of wild type group. Moreover, there Was a significantly decreased trend for swim time and swim distant in G-CSF group(P＜0.01). There Was no effect of block or treatment on swim speed, and no treatment by block interaction. In the Spatial retention trial, both quadrant time and platform crossings,There was no statistical difference between wild type group and G-CSF group(P<0.05) (Figurel-4).
2. HE and Nissl staining show a clear structure of hippocampus neurons.The cell nucleus structure is normal.The Nissl's bodies in the intracytoplasm were abundant.The chromatins of cell nucleus were distributed equally. Hippocampus neurons of Alzheimer's disease mice model were injuried.Swelled neurons and broken neurons were observed and they ranked chaos in the sections.The Nissl's bodies in the intracytoplasm and survival neurons were reduced compared with control groups (P<0.05).The structure of hippocampus neurons were similar as the wild type groups (Figure5,6,Tablel,2)
Conclusions
1. The cognition function of Alzheimer's disease mice model was decreased.
2. The cognition function of Alzheimer's disease mice model was improved after the administration of G-CSF.
3. G-CSF administration was benefited for the cognitive function of Alzheimer's disease mice model.
Part 2 Influence of G-CSF on the neuroinflammation in the brain of Alzheimer's disease mice model
Objective
Investigate the influence of G-CSF on the neuroinflammation in the brain of Alzheimer's disease mice model.
Methods:
1. Alzheimers disease mice model and wild mice were divided into G-CSF group、control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of IL-1βand TNF-a in mice's frontal lobe cortex was detected by immunofluorescence, and observe the impact of G-CSF on their expression.
Results
1. Analysis result of image showed that the relative immunofluorescence intensity levels of IL-1βin the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P＜0.05) (Figurel,2). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figurel,.2). There is significate difference bwteen three groups (P<0.05).
2. Analysis result of image showed that the relative immunofluorescence intensity levels of TNF-a in the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P<0.05) (Figure3,4). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figure3,4). There is significate difference bwteen three groups (P<0.05).
Conclusions
1. It was confirmed in my research that neuroinflammation exist in the brain of Alzheimer's mice model.
2. MDL-28170 protects hippocampal neuron after pilocarpine-induced seizure against PCD through the inhibited effect onμ-calpain.
3. The administration of G-CSF attenuate neuroinflammtion in Alzheimer's mice model through repress the expression of inflammatory factors IL-1βand TNF-a. It protected the cortical neurons from neuroinflammation of Alzheimer's disease mice model.
Part 3 Influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model
Objective
Investigate the influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model. Discuss the critical effect of a7 nAChR on the cholinergic anti-inflammatory pathway.
Methods:
1. Alzheimers disease mice model and wild mice were divided into G-CSF group、control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of a7 nAChR in mice's frontal lobe cortex was detected by immunohistochemistry (IHC), and observed the impact of G-CSF on their expression.
3. Mice were sacrificed at different time-points. Their brains were obtained and stored at-80℃.
4. The proteins expression of a7 nAChR was detected by Western blotting and the effects of G-CSF on their expression were observed.
Results
1.Analysis result of image showed that density values ofα7nAChR in G-CSF day 7 group, G-CSF day 14 group, G-CSF day 28 group, G-CSF day 56 group were higher statistically than that of control group and wild type group(P<0.01 or 0.05). There is significant difference between the three groups (P<0.05) (Figurel,2).
2.Comparison of protein levels ofα7nAChR:The protein levels of a7nAChR protein in G-CSF group increased significantly compared to that in control group(P<0.01).There is significant difference between the three groups (P<0.05) (Figure3,4).
Conclusion
1.α7 nAChR expressed generally on the front lobe cortex of mice's brain.
2. The expression of a7 nAChR on the front lobe cortex of Alzheimer's mice model's brain was significately depressed.
3. The administration of G-CSF could increase the expression of a7 nAChR on the brain of Alzheimer's mice model in different time points. It has the function of neuroprotection.
Part 4 Influence of G-CSF on the expression NF-kB in the brain of Alzheimer's disease mice model and study of inflammation mechanisms
Objective
Investigate the influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model. Discuss the critical effect of a7 nAChR on the cholinergic anti-inflammatory pathway.
Methods
1. Alzheimers disease mice model and wild mice were divided into G-CSF group,control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.
2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of NF-κB in mice's frontal lobe cortex was detected by immunofluorescence, and observed the impact of G-CSF on their expression.
3. Spearman correlation analysis was used to analysis the relationship of the expression ofα7 nAChR and NF-κB at different time points.
Results
1. Analysis result of image showed that the relative immunofluorescence intensity levels of NF-κB in the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P<0.05) (Figurel,.2). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figurel,.2). There is significate difference bwteen three groups (P<0.05).
2. Result of Spearman correlations analysis showed that there was significant negative correlations betweenα7 nAChR and NF-κB expressions (7d:rho=-0.73, p=0.007; 14d:rho=-0.83, p=0.008; 28d:rho=-0.89, p=0.02).
Conclusion there is an inverse correlation between the expression ofα7 nAChR and NF-κB in the brain of Alzheimer's disease mice model after the treatment of G-CSF. This finding suggests that G-CSF significantly attenuates the expression of proinflammatory cytokines by increasing the expression ofα7 nAChR in the brain of APP transgenic mice.
引文
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