【作者】 吕兰海；

【导师】 李继承； 姚大卫；

【作者基本信息】 浙江大学 ， 细胞生物学， 2007， 博士

【摘要】 脑老化是人们在享受长寿时不可避免的事件。大量的报道指出，随着年龄的增加，大脑内的神经元数量逐渐减少，而神经胶质细胞的数量和体积逐渐增加，这可能是脑老化的基础。衰老是引起很多疾病的诱因，阿尔茨海默氏病（Alzheimer’s disease，AD）就是最常见的一种衰老相关的疾病。目前已经证实，β淀粉样蛋白沉积在细胞外形成的老年斑和过磷酸化的tau蛋白在细胞内形成的神经纤维缠结是AD最主要的病理学特征。神经胶质细胞是中枢神经系统含量最多的细胞，特别是星形胶质细胞占脑内细胞数量的25％，是哺乳动物脑内分布最广泛的一类细胞。星形胶质细胞广泛参与神经系统的许多功能，如调节和维持细胞外环境的稳定，吸收和释放神经递质和神经调质，调节突触传递和神经元刺激性，为神经元提供营养、能量代谢物质和神经递质前体物，参与脑内自由基的捕获，指导发育过程中神经元的迁移，参与神经系统的免疫和炎症反应。目前已知星形胶质细胞，特别是年轻或未成熟的星形胶质细胞具有很好的支持神经元生长的能力。然而，随着衰老进展，星形胶质细胞的这种能力逐渐降低。尤其是成熟的星形胶质细胞已经不能提供神经元再生所必需的表面分子、细胞外基质分子和神经营养因子。人们通常认为，神经元的丢失是引起衰老性疾病患者记忆力丧失的主要原因。因此，神经元的研究一直是衰老及其相关疾病研究的中心。在大脑中，神经胶质纤维酸性蛋白（GFAP）是正常成年中枢神经系统星形胶质细胞主要的中间纤维蛋白，vimentin作为另一种中间纤维蛋白，其表达水平却很低。当大脑受到缺血、外伤或神经退行性变时，星形胶质细胞的突触就会大量增生，GFAP和vimentin表达上调，这种增生的过程常常伴有许多蛋白表达的改变。胶质增生常被看作一种对大脑损伤的保护作用，然而过渡增生可直接导致星形胶质细胞死亡。胶质细胞的增生反应在老化过程的作用往往被人们忽视，因为人们普遍认为衰老过程中胶质细胞的激活是次于神经元的老化。Finch（2003）的研究指出，在衰老未出现明显的可诊断病理前，星形胶质细胞就已经被激活。我们最近的研究证实，在正常衰老的SAM R1小鼠大脑海马区星形胶质细胞GFAP表达显著增加的，具有衰老倾向的老年SAM P8小鼠GFAP表达水平明显高于同龄对照SAM R1小鼠表达水平。目前已知，星形胶质细胞和神经元之间存在广泛的突触联系。神经元激活、释放的神经递质可以刺激星形胶质细胞内钙离子内流增加，激活星形胶质细胞。活化的星形胶质细胞也可以表达表面分子，释放许多亲神经因子和细胞活素。星形胶质细胞释放的这些物质又可以反馈性地调节神经元的递质释放或直接作用于突触后神经元引起抑制或激活效应。衰老过程中星形胶质细胞的过度增生，必然引起胶质细胞对神经元的正常调节功能改变。正常情况下，β-淀粉样前体蛋白（APP）和tau蛋白主要分在神经元内，在星形胶质细胞也可以低水平表达。然而，Miyazono等（1993）研究指出，在AD和其他神经系统退行性疾病，常可出现tau免疫反应阳性增生的星形胶质细胞。Schultz等（2000）的研究指出，在衰老的狒狒星形胶质细胞GFAP表达水平明显增加，并且伴有tau蛋白的聚积。星形胶质细胞产生的APP和tau蛋白在细胞老化过程中很可能会影响星形胶质细胞的功能。迄今为止，尚未发现APP和tau蛋白对原代培养的星形胶质细胞的研究报道。衰老加速小鼠（SAM）最初是由Takeda等（1981）年通过对AKR/J系小鼠杂交产生的，是一种衰老加速的小鼠动物模型。Miyazaki报道（2003），目前已有13株SAM小鼠建立，其中9株为衰老倾向的小鼠，4株为衰老阻抗的小鼠。衰老倾向小鼠（SAMP）通常表现为具有较短的生命周期，增加的淀粉样变性，线粒体功能失常，以及学习和记忆能力的不足。而衰老加速阻抗小鼠（SAMR）通常表现出正常衰老的特征。因此，SAM小鼠是研究在衰老过程中星形胶质细胞功能的较理想动物模型。目前，β-淀粉样蛋白在衰老的星形胶质细胞的研究很少，也无tau蛋白对体外培养的星形胶质细胞的研究报道。本实验我们首先建立了两分别来自SAM R1和P8新生小鼠大脑皮质的原代培养星形胶质细胞，模拟衰老过程中的星形胶质细胞。继而以这两细胞作为实验对象，研究了不同浓度的过氧化氢、Aβ1-42和tau蛋白对不同分化成熟的星形胶质细胞的作用，探讨星形胶质细胞在衰老过程中的功能变化。第一部分星形胶质细胞的建立及细胞增殖能力检测目的：建立两分别来自衰老倾向小鼠SAM P8和R1新生鼠大脑皮质星形胶质细胞，并检测两细胞的增殖能力。方法：（1）采用原代培养方法从新生（1-3天）SAMP8和R1小鼠大脑皮质区获得星形胶质细胞，接种于无菌细胞培养瓶，于37℃5％CO₂孵箱培养并观察细胞的生长状况。于第7-8天在200rmp/min转速下振摇纯化细胞8h，弃去悬浮细胞并换新的培养基继续培养，直至进行下一步实验；（2）采用免疫细胞化学检测GFAP蛋白在星形胶质细胞中的表达，并检测获得的两星形胶质细胞的纯度；（3）MTT还原实验检测两细胞的增殖能力。结果：（1）光镜下，接种3天后细胞逐渐展开，伸出突起，但还没有形成生发中心；体外培养5天时，已经出现较多的扁平细胞和多角形星形胶质细胞。7天后，星形胶质细胞开始大量增殖，并形成较多的生发中心。在14-16天时，明显可见细胞出现融合。体外传4代后，一些星形胶质细胞开始失去增殖能力，表现出“上皮样”形态。（2）绝大多数细胞（约95％）呈现强或弱阳性GFAP染色，细胞可呈现星形、单极和多角形等多种形态。（3）MTT实验结果表明，传代接种72小时后，开始进入明显的增殖期，与24小时比具有显著性差异（p＜0.05），而后进入指数增长期。至144小时，细胞开始融合、出现接触抑制，细胞增殖开始减慢。两细胞间比较，SAM R1株小鼠细胞在72小时明显快于SAM P8株小鼠细胞，这种趋势一直维持到细胞出现融合。两小鼠星形胶质细胞均有较强的增殖能力。结论：我们首次原代培养了两来自具有不同衰老程度的小鼠（SAMR1和P8）新生鼠大脑皮质星形胶质细胞。SAMR1和P8新生鼠大脑皮质星形胶质细胞在体外培养时可能具有不同的增殖能力。第二部分过氧化氢对两星形胶质细胞的作用目的：研究并比较不同浓度的过氧化氢（H₂O₂）对两星形胶质细胞（SAM P8和R1）的作用。方法：不同浓度的过氧化氢（H₂O₂）（0，100，200，400μM）分别处理两细胞1小时或4小时后，扫描电子显微镜（SEM）观察过氧化氢对两星形胶质细胞的形态学影响；MTT实验检测两细胞还原能力；碘化丙啶染色荧光镜下检测细胞存活；末端脱氧核苷酸转移酶介导的dUTP缺口标记技术（TUNEL）的染色方法原位检测细胞凋亡；免疫组织化学检测细胞乳酸脱氢酶活性；Western blot检测过氧化氢对星形胶质细胞胶质纤维酸蛋白（GFAP）、超氧化物歧化酶（SOD）及凋亡相关蛋白caspase-3和Bax的表达水平。结果：（1）SEM下显示两胶质细胞通常表现出星形状，并且细胞表面展现出大量短的纤毛，细胞表面可见有长的突起伸展，并且与其他细胞建立联系，这些细胞通常有棒状末端，较多的纤毛和侧突。在低剂量过氧化氢（100μM）处理R1株小鼠细胞时，在1-4小时没有明显的效应。随着过氧化氢浓度的增加（200μM）并且作用较长的时间（4h）时，大约有30％的纤毛丢失，侧突也变短。用更高浓度的过氧化氢（400μM）处理4小时后，绝大多数纤毛消失。相似的形态学改变同样在P8株小鼠培养的细胞中也可见到。随着过氧化氢浓度的增加，纤毛逐渐变少，侧突变少、缩短。两细胞相比，P8株小鼠培养的细胞纤毛的丢失明显比R1株小鼠细胞严重，特别是在200μM处理4小时。细胞破裂后，两细胞各剂量组及其未处理的正常细胞，细胞内均可见细纤维（微管）和小的球形颗粒。R1和P8两小鼠来源的细胞用低剂量（100μM）过氧化氢处理较长时间（4h）时，可见微管和球形颗粒存在。用高剂量的过氧化氢（400μM）处理较短的时间（1h），可引起球形颗粒物明显增加，然而较高的剂量（200μM）作用较长时间（4b）时，可明显减少球形颗粒物，并且引起微管凝集和增粗。（2）经不同浓度的过氧化氢处理后，两细胞的MTT还原能力与对照相比均显著降低（p＜0.05）。以R1对照组细胞MTT还原能作为100％，P8细胞的MTT还原能力仅为81.4±7.4％，具有统计学意义（p＜0.05）。（3）碘化丙啶试验表明，两细胞经过氧化氢处理后死亡率随浓度增加而增高，两未加过氧化氢处理细胞组细胞死亡率分别为：R1细胞为11±2.53％，P8细胞为10.6±2.22％。与未用过氧化氢处理的对照组（control）相比，在200μM和400μM浓度时过氧化氢可显著增加细胞死亡（p＜0.05）。在200μM浓度时P8株小鼠细胞的死亡率（27.46±2.61％）明显高于对应的R1株小鼠细胞的死亡率（18.1±1.78％）（p＜0.05）。（4）随着过氧化氢浓度的增加，在较高的浓度400μM时，P8株小鼠细胞TUNEL原位凋亡率明显高于R1株小鼠细胞（p＜0.05）。（5）过氧化氢处理后两细胞（P8和R1）乳酸脱氢酶标记阳性率随着浓度的增加逐渐增加，与未加过氧化氢组相比在较高浓度（400μM）时两细胞均有显著性增加（p＜0.05）。在较高浓度（400μM）时P8株小鼠细胞死亡率高于R1株小鼠细胞（p＜0.05）。（6）随着过氧化氢处理浓度的增加，P8和R1两小鼠来源的细胞其特异性表达胶质纤维酸蛋白的表达水平具有相似的降低趋势。而且，P8各剂量组胶质纤维酸蛋白表达水平均显著高于R1小鼠来源的细胞组表达水平；在较高的浓度（400μM）时两细胞超氧化物歧化酶（SOD）表达水平显著增加（p＜0.05）。P8小鼠细胞表达水平明显高于相应的R1小鼠细胞表达水平（p＜0.05）；与对照组相比，经400μM过氧化氢处理后caspase-3的表达水平在两细胞均有显著增加（p＜0.05）。在400μM时，R1株小鼠细胞caspase-3的表达水平明显低于P8小鼠细胞的表达水平（p＜0.05）；经逐渐增加浓度的过氧化氢处理后，两细胞Bax的表达水平均有所增加，尤其是R1株小鼠细胞。与相应的对照（不加过氧化氢组）相比，P8株小鼠细胞组间没有统计学差异。用400μM过氧化氢处理后，P8株小鼠细胞Bax的表达水平明显低于R1株小鼠细胞的表达水平（p＜0.05）。结论：两分别来自SAM P8和R1新生小鼠大脑皮质区的星形胶质细胞用过氧化氢处理后，两细胞死亡率具有显著性差异。在损伤后，两细胞具有相同的形态学改变如微绒毛和侧突丢失。高浓度过氧化氢（400μM）短时间处理细胞（1小时）能增加细胞的合成；而较高浓度过氧化氢（200μM）长时间处理细胞（4小时）能降低细胞内合成，引起微管聚合。过氧化氢处理后两细胞可以有相似的效应：MTT还原能力和胶质纤维酸蛋白表达下降，碘化丙啶检测细胞死亡增加，TUNEL染色方法原位检测细胞凋亡增加，超氧化物歧化酶、切冬酶-3和Bax表达上调。过氧化氢400μM处理时，P8细胞和R1细胞反应具有显著性差异（p＜0.05），表明P8和R1两细胞在对抗氧化应急时可能有不同的后果，并提示在高浓度过氧化氢刺激时能明显减弱P8星形胶质细胞对神经元丢失的保护作用，目前机制尚不清。老化程度不同的星形胶质细胞对过氧化物解毒能力改变在中枢神经系统衰老和衰老相关的发病过程，可能有重要的作用。第三部分Aβ（1-42）和Tau蛋白对两星形胶质细胞的作用目的：探讨并比较Aβ（1-42）和tau蛋白单独或联合处理对两星形胶质细胞的作用。方法：用不同浓度的Aβ（1-42）（1μM和5μM）、tau蛋白（100nM）及两者物质的混合物Aβ（1-42）1μM/5μM+tau蛋白（100nM）及不含Aβ和tau蛋白的DMEM/F-12无血清培养基（对照组），分别处理两星形胶质细胞（SAM P8和R1）24小后，用免疫细胞化学分别检测蛋白激酶C（cPKC）、己糖激酶（Hexokinase）、乳酸脱氢酶（LDH）、Aβ前体蛋白（APP）、NMDA受体、GFAP在两细胞的表达，用Western blot分别检测GFAP、乳酸脱氢酶（LDH）、切冬酶-3（caspase-3）、N-甲基-D-天冬氨酸（NMDA）受体、己糖激酶（Hexokinase，HXK）、蛋白激酶C（cPKC）、Aβ前体蛋白（APP）、S100β蛋白分别在两细胞中的表达。应用激光共聚焦显微镜观察Aβ（1-42）和tau蛋白处理后两细胞GFAP蛋白和微管蛋白（Tubulin）的共表达。结果：（1）cPKC活性检测：与R1细胞相比，体外培养的P8细胞具有较低的cPKC活性。tau蛋白单独或与Aβ（1-42）联合处理24小时后可明显增强P8细胞cPKC活性，与相应的对照比差别显著（p＜0.05）。tau100nM能显著增加P8细胞cPKC活性，与相应的R1细胞比差异显著。Aβ单独作用也可增加R1细胞cPKC活性，但与tau蛋白联合作用后无明显效应。（2）HXK活性检测：与R1各组细胞（包括对照组）相比，体外培养的P8细胞均有较低的HXK活性。Aβ和tau蛋白单独处理24h后均可以增高两组细胞HXK活性，并且在Aβ5μM时，P8细胞HXK活性明显低于相应的R1细胞。Aβ和tau蛋白联合处理，可增高P8细胞HXK活性，高剂量Aβ（5μM）与tau蛋白联合处理可降低R1细胞HXK活性。（3）LDH活性检测：与相应的R1细胞比，体外培养的P8细胞均由较低的LDH表达水平（p＜0.05）。Aβ（1-42）与tau蛋白单独或联合处理24小时后，均增高两组细胞LDH的表达水平，与相应的对照组比较具有统计学意义（p＜0.05）。（4）APP表达水平检测：体外培养的两细胞均可以表达APP蛋白。与相应的R1细胞相比，体外培养的P8细胞具有较低的APP表达水平。Aβ（1-42）和tau蛋白单独和联合处理均可以下调APP表达。特别是Aβ5μM和tau蛋白联合处理可显著降低APP表达水平，与相应的对照差别显著。（5）NMDA受体表达检测：体外培养的SAM R1细胞和P8细胞可以表达NMDA受体。与R1细胞相比，体外培养的P8细胞具有较低的NMDA受体表达水平。Aβ（1-42）和tau蛋白单独和联合作用时，均可以增加P8细胞NMDA受体表达水平，并且tau蛋白单独或与Aβ（1-42）联合处理时显著高于相应的对照细胞。而在相应的R1细胞，Aβ（1-42）单独作用可以增加NMDA受体表达水平，但tau蛋白单独或与Aβ（1-42）联合处理时降低NMDA受体表达水平。（6）切冬酶-3表达检测：与相应的R1细胞比，体外培养的P8细胞具有较低的caspase-3的表达水平。Aβ（1-42）与tau蛋白单独或联合处理均可以增加R1细胞caspase-3的表达水平，特别是在tau蛋白100nM单独或与Aβ（1-42）共处理时，可显著增加caspase-3的表达。Aβ（1-42）与tau蛋白单独或联合处理也可以增加P8细胞caspase-3的表达水平。与相应的R1细胞比，P8细胞在tau蛋白100nM单独或与Aβ（1-42）共处理时均有较低的caspase-3的表达。（7）S100β蛋白表达检测：与相应的R1细胞比，体外培养的P8细胞在Aβ（1-42）和tau蛋白处理前后均有较低的S100β蛋白表达水平（p＜0.05）。Aβ（1-42）和tau蛋白单独或联合处理后R1细胞S100β蛋白表达水平均显著增加，与相应的对照比差别显著。Aβ（1-42）和tau蛋白单独作用均可以显著降低P8细胞S100β蛋白表达水平，但两者共处理时却可以显著增加S100β蛋白表达水平（p＜0.05）。（8）微管蛋白（Tubulin）和胶质纤维酸蛋白（GFAP）共表达检测：Aβ（1-42）和tau蛋白单独或联合处理后均可不同程度上调两细胞GFAP表达，特别是在高浓度Aβ单独或联合与tau共作用时与相应的对照比差别显著（p＜0.05）。与相应的R1细胞相比，tau蛋白单独处理或与Aβ（1-42）联合作用后P8细胞均具有较低的GFAP表达水平。应用激光共聚焦显微镜观察Aβ（1-42）和tau蛋白处理细胞后，可见Aβ（1-42）单独或与tau蛋白共处理使两星形胶质细胞形态明显受损，细胞呈现出破碎现象。而tau蛋白处理过的细胞虽然也可以影响胶质纤维酸蛋白和微管蛋白的表达，但却不影响细胞的形态学表形。结论：首次应用Aβ（1-42）和tau蛋白对两不同分化成度的细胞（SAMR1和P8）比较研究，发现Aβ（1-42）与tau蛋白对两细胞（SAM R1和P8）的cPKC、HXK、LDH、APP、NMDA receptor、Caspase-3、S100β和GFAP等表达能产生影响，并且在两细胞可产生不同后果，为研究衰老过程中星形胶质细胞的功能改变提供了依据。更多还原

【Abstract】 Aging is an inevitable event when we enjoy increased longevity. Many reports pointed out that the decreased numbers of neurons and the increased numbers and volumes of glia may be the main basic changes of brain aging. Aging is associated with a dramatic increase for the risk of many aging related neurodegenerative disorders, such as Alzheimer’s disease （AD）. Currently, it was confirmed that amyloid β and tau protein, the major components of senile plaques and neurofibrillary tangles, respectively, had been considered as central mediators of the pathogenesis of Alzheimer’s disease.Glia greatly outnumber other cells in our central nervous system, especially astrocytes comprised 25% percent of all the brain cells. Astrocytes can widely participate in the activities in our brain, such as the maintenance and regulations of the extracellular environment; provision of nutrients; energy substrates and neurotransmitter precursors; free radical scavenging; guidance of neuronal migration during development; and immune/ inflammatory functions. To our knowledge, astrocytes, especially young or immature astrocytes can strongly support neurons. During aging, astrocytes have the decreased capabilities to do so. In particular, aged astrocytes could not provide the necessary cell surface molecules, extracellular matrix molecules and neurotrophic factors.Commonly, the loss of neurons was regarded as the main cause of the memory deficiency in aging related diseases, so the study of the neurons was always the focus in aging and aging related neurodenegerative disorders. Glia fibrillary acid protein （GFAP） is the major intermediate filament of astrocytes in adult nervous system, vimentin as another intermediate filament has the low level of expression. The hallmark of reactive gliosis in CNS ischemia, trauma or in neurodegeneration is characteristic hypertrophy of cellular processes of astrocytes and upregulation of GFAP and vimentin, accompanying alterations in expression of many proteins.The hypertrophy of astrocytes plays a protective role against the development and progression of brain injury, however sometimes the hypertrophy is lethal to astrocytes. Theeffects of the astrocytic hypertrophy during normal aging were often neglected because the astrocytic activation was secondary to neuron loss. Finch （2003） recently had shown that astrocytes were already activated without the presence of obvious, diagnosable pathology during aging. Our current data also indicated that significant age related increases in the numbers of astrocytes and the expressions of GFAP in the hippocampus of aged SAM mice. As well, the expression levels of GFAP in aged SAMP8 mice were significantly greater than those in matched aged SAMR1 mice.Currently, it was established that there were wide intercommunications between astrocytes and neurons. Activated neurons can release many neurotransmitters to stimulate astrocytes to take up Ca²⁺, which plays an important role in activation of astrocytes. Activated astrocytes also can express many surface molecules; release a lot of neurotrophic factors and cytokinases. Such active substances can reinforce the feed loop of astrocytes onto neurons to modulate the release of the neurotransmitters or directly to inhibit or activate the postsynaptic neuron activities. The modulation of neural functions was inevitably influenced by the astrocytic hypertrophy during aging.Normally, the β-amyloid precursor protein and tau protein were mainly expressed in neurons, and could also be present at low level in astrocytes. However, Miyazono et al （1993） had confirmed that there were many tau immunopositive astrocytes in the brain of AD and other neurodegenerative diseases. As mentioned above, the metabolic turnover of GFAP was increased in aged glia. Accumulations of tau, although not neurofibrillary tangles （NFT）, have also been found in astrocytes and oligodendrocytes of aged baboons. The expressed β-amyloid precursor protein （APP） and tau protein in aged glia might affect astrocytic functions. Up to date, there were limited reports about these effects induced by APP and tau protein in primary cultured astrocytes.Senescence-accelerated mouse （SAM） strains were originated from the ancestral AKR/J strains as established by Takeda in 1981, and were seen as a murine model for accelerated aging. At present, there are at least 13 lines of SAM: nine senescence-prone strains and four senescence-resistant strains. The senescence-accelerated-prone mice （SAMP） exhibit accelerated aging with a shortened life span, increased amyloidosis, mitochondrial dysfunction, as well as learning and memory deficits. Senescence-accelerated-resistant mice （SAMR） exhibit normal aging features. Therefore, the SAM mice are good models to study astrocytes during aging.Until now, the studies about β amyloid in aged astrocytes were very limited, and also few research works about tau protein in in vitro astrocytes were reported. In our present study, two strains of primary cultured astrocytes were developed separately from the cortex of SAM P8 and R1 neonatal mice （1-3 days） to model the astrocytes during aging. In the following experiments, the cell samples prepared were treated separately by different concentrations of hydrogen peroxide, Amyliod β1-42 and tau protein, and the effects in two lines of astrocytes were investigated and compared.Part Ⅰ The establishment of two astrocyte stains and the cell proliferation assayAims: to establish the two astrocyte strains from the cortex of SAM P8 and R1 neonatal mice, and to investigate their proliferative capabilities. Methods: （1）the primary astrocytes were prepared from the cortex of SAM P8 and R1 neonatal mice （1-3 days） by dissection and tissue culture methods. Then the acquired cell suspensions were plated into sterile flasks, and cultured under 37°C 5% CO2 environment. During the culture period, the cells were observed and photographed to compare the proliferation of the two astrocytes. At 7-8 days after in vitro subculture, the cells plated into flasks were purified by shaking at 200 rpm 37℃ for 8 hours to dislodge the contaminated cells, such as microglia and other cells, then the astrocytes were cultured for another week before subsequent experiments; （2）the purity of two astrocytes were assayed by immunocytochemical assay using the antibody of astrocytic marker protein GFAP;（3） Cell proliferation was also tested by 3-（4,5-dimethylthiazol-2-yl）-2,2-diphenyltetrazolium bromide （MTT） reduction assay. Results: （1） Under light microscopy, the cells plated at 3 days after in vitro subculture began to extend their side processes, but no concentric reparatitions; after 5 days after in vitro subculture, the proliferating centers were observed and surrounded by flat and polygonal astrocytes; at 7 days after in vitro subculture, the astroglial populations began to increase strongly with concentric repartitions; at about 14-16 days in vitro （DIV）, the astroglial culture was definitely confluent; after 4 passages of subculture, some astrocytes lost their abilities to proliferate and adopted an "epithelioid" morphology. （2）the cells plated onto 11 mm round coverslips pretreated by poly-dl-ornithine were stained by GFAP immunocytochemistry and photographed. About 95 percent of total astrocytes were GFAP positive although some cells were stained dim, and astrocytes exhibit stellate, unipolar or polygonal morphology. （3）At 72 hours after subculture, the cells strongly proliferated and entered into exponential phrase. After 7 days after subculture, the cells become confluent and started to stop dividing. Compared the proliferation of astrocytes originated from SAM P8 at 72h after subculture, the SAM R1 derived astrocytes had a higher proliferative ability. Conclusions: Through primary cultures obtained from the cortex of SAM P8 and R1 neonatal mice （1-3 day）, we had established highly enriched populations of astrocytes that were about 95% purity as judged by immunophenotypical expression of GFAP, they all had strongly proliferative function.Part Ⅱ The effects induced by hydrogen peroxide on two strain astrocytesAims: to investigate and compare the effects induced by different concentrations of hydrogen peroxide （H₂O₂） （0,100,200,400μM） in two SAM （P8 and R1） derived astrocytes.Methods: two group astrocytes were treated by different concentrations of H₂O₂ （0, 100, 200, 400μM） for one or four hours, scanning electron microscopy（SEM） was used to observe the astrocytic morphologies; MTT assays were performed to test the reduction ability; cell death was assay by propidium iodide （PI） under fluorescence microscope; cell apoptosis was assayed by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling（TUNEL） assay; Cell necrosis was assayed by lactate dehydrogenase（LDH） immunocytochemical assay; the expressions of GFAP, superoxide dismutase （SOD）, Caspase-3 and B-cell lymphoma 2-associated protein （Bax） were detected by Western blot. Results: after treatments by different concentrations of H₂O₂ （0, 100, 200, 400 μM）,our SEM results illustrated that the cultures of astrocytes from both R1 and P8 strains were usually star shaped with numerous short cilia on the cell surfaces. Long projections also appeared from all around the culture cells, making contacts with other culture cells. These cells usually had club endings. Exposure of R1 strain culture astrocytes from one to four hours of H₂O₂ at a dosage of 100μM showed little damage effect. The astrocytes maintained an abundant amount of cilia and side projections and with increasing dosage （4 hours of 200μM）, roughly 30% of the cilia were lost and side projections became short. At even higher dosage （4 hours of 400μM）, most cilia were lost although some side projections were still evident. A similar situation occurred in the P8 strain cultured astrocytes. With increasing dosage, the cilia became less and side projections decreased and shortened. The only difference was that the loss of cilia was more vigorous in the P8 cells than in the R1 cells, with cilia decreasing in number rapidly at a dosage of 200μM for 4 hours. When the cells were fractured, in both the control untreated R1 and P8 cells, thin fibers （microtubules） with small globules were observed internally. In both R1 and P8 astrocytes, after 4 hours treatment by low dosage （100μM）, the microtubules and globules were still present. Treatment of a high dosage （400μM） of H₂O₂, within a shorter period of an hour resulted in an increase of globular material, while a high total dosage （200μM in 4 hours） eliminated globular material and caused coagulation and thickening of microtubules. （2） For the MTT assays, the percentage of MTT reduction decreased significantly （p<0.05） with increasing H₂O₂ concentrations as compared to that of the controls （cultures not exposed to H₂O₂） derived from both P8 and R1 strains. Further, the MTT reduction of the P8 control was 81.4±7.4% when compared to the R1 control as 100%, and the difference was statistically significant （p<0.05）. （3） Overall cell death in astrocytes derived from both P8 and R1 mice was assessed by the PI assay. Results showed cell death percentage increased with increasing H₂O₂ concentrations. With no addition of H₂O₂, the cell death in R1 was 11±2.53% and P8 was 10.6±2.22%. As compared to the cell death percentage of the above control （zero H₂O₂ concentration）, the increases at 200μM and 400μM of H₂O₂ treatment were statistically significant （p<0.05）. At 200μM of H₂O₂, the difference between P8 samples （18.1±1.78%） and R1 samples （27.5±2.61%） was statistically significant （p<0.05）. （4） The extent of apoptosis in the astrocyte culture was visualized by TUNEL in situ hydridization. The percentage of astrocytes stained positive for TUNEL increased withincreasing H₂O₂ concentrations in cultures derived from both P8 and R1 strains, and the increases were statistically significant （p<0.05） as compared to the controls （cultures not exposed to H₂O₂）. （5） The percentage of cell stained positive with LDH increased with increasing H₂O₂ concentrations in cultures derived from both P8 and R1 strains. The increases were statistically significant （p<0.05） from controls （cultures not exposed to H₂O₂） when the P8 and R1 cultures were treated with 400 μM of H₂O₂. The differences between P8 samples and R1 samples were statistically significant （p<0.05） only when treated with 400 μM of H₂O₂. （6） When treated with increased concentrations of H₂O₂, the decreases in GFAP levels were statistically significant （p<0.05） as compared to the controls （cultures not exposed to H₂O₂）. In addition, there were large and statistically significant （p<0.05） differences in GFAP levels between P8 and R1 samples treated with each of the H₂O₂ concentrations as well as between controls; SOD levels shown by Western blot increased with H₂O₂ concentrations in cultures derived from both P8 and R1 strains. Again, only when treated with a H₂O₂ concentration of 400 μM, were the increases in SOD levels statistically significant （p<0.05） as compared to the controls （cultures not exposed to H₂O₂）. Likewise, the difference between P8 samples and R1 samples was statistically significant （p<0.05） when treated with 400 μM of H₂O₂; in both P8 and R1 samples, Caspase-3 levels increased significantly （p<0.05） when treated with 400 μM of H₂O₂ as compared with the controls （cultures not exposed to H₂O₂）. In addition, the differences between P8 samples and R1 samples was statistically significant （p<0.05） at this concentration of H₂O₂; Bax expression level were increased in all groups treated by H₂O₂, especially in R1 samples. There were no statistical significant differences in P8 samples as compare with the controls （cultures not exposed to H₂O₂）. The difference between P8 samples （1.122±0.224） and the corresponding R1 samples （1.525±0.182） was statistically significant （p<0.05） when treated with 400 μM of H₂O₂. Conclusions: A mild but statistically significant difference was observed in the numbers of cell death between R1 and P8 cells after H₂O₂ treatment. Cellular changes were equivalent in both strains after injury, including loss of cilia and side projections. High total dose of H₂O₂ treatment （e.g. 400μM for only one hour） caused increased cellular synthesis, while high total dose of H₂O₂ treatment （e.g. 200μM for four hours） downregulated in intracellular synthesis and caused coagulation of microtubules. Our results showed that the oxidative stress had similar effects in both strains of astrocytes: decreases in MTT recution and GFAP levels and increases in cell death by PI staining, TUNEL, LDH staining and the expression levels of SOD, Caspase-3 and bax. At a H₂O₂ concentration of 400 μM, the differences of the above parameters between P8 cultures and R1 cultures were statistically significant （p<0.05）. This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a H₂O₂ concentration of 400 μM remains elusive. Different age astrocytes may play an alternative role in detoxification of toxicants, and may exert an important function in CNS aging and aging related neurodenegerative disorders.Part Ⅲ The effects Induced by Aβ（1-42） and Tau protein on two strain astrocytesAims: to investigate and compare the different effects induce by Aβ（1-42） and tau protein on two strain astrocytes（SAM P8 and R1）. Methods: two strain astrocytes （SAM P8 and R1） prepared as above were separately treated by Aβ （1-42）（1μM or 5μM）, tau protein （100nM） and mixed solutions [Aβ（1-42） 1μM or 5μM + tau protein 100nM] or DMEM/F-12 medium without fetal bovine serum （FBS） （control group） for 24 hours, then the following experiments were performed. Using immunocytochemical assay, the expression level of protein kinase c（cPKC）, hexokinase, LDH, Amyloid β precursor protein （APP）, N-methyl-D-aspartate （NMDA） receptor and GFAP were investigated; using Western blot method the expression levels of cPKC, GFAP, LDH, Caspase-3, NMDA receptor, hexokinase, APP and S100β protein were separately detected. The coexpression of GFAP and tubulin in astrocytes treated by Aβ （1-42） and tau protein were analysed by confocal laser scanning microscopy （CLSM）. Results: （1） Compared with the corresponding R1 astrocytes, the In vitro P8 astrocytes had low expression levels of cPKC. By the treatment of single tau protein or combined treatment of Aβand tau protein for 24h, the expressions of cPKC in P8 astrocytes could be significantly increased; there were significant differences between the treated groups and the control groups. Compared with the expression of cPKC in R1 astrocytes, tau protein （100nM） could significantly enhance the expressions of cPKC in P8 astrocytes. Aβ solely enhanced the the expressions of cPKC in R1 astrocytes, but there were no obvious effects by combined treatments of Apand tau protein. （2） compared with the corresponding R1 astrocytes （including the control group）, In vitro P8 astrocytes had lower expression levels of hexokinase. Aβ or tau could solely significantly enhance the expression levels in both R1 and P8 astrocytes. When treated at the dosage of Aβ5μM, the expression of HXK in P8 astrocytes could be significantly decreased compared with that in R1 astrocytes. The combined treatments of Aβand tau protein could significantly enhance the expressions of HXK in P8 astrocytes, and decreased the expressions in R1 astrocytes, especially by the treatment of Aβ（5μM）and tau protein （100nM） .（3） Compared with the corresponding R1 astocytes, in vitro P8 astrocytes had lower expression level of LDH （p<0.05）. Compared with the expression of each control group, there were significantly increased expressions of LDH in two astrocytes （R1 and P8） with 24 hour’s sole or combined treatment by Aβ and tau protein（p<0.05）.（4） Both in vitro astrocytes could express APP. Compared with the corresponding R1 astrocytes, in vitro P8 astrocytes had lower expression levels of APP. Especially the combined treatment of Aβ5μM and tau （100nM） could significantly decrease the expressions of APP in both astrocytes（p<0.05）.（5） The expressions of NMDA receptors were obviously detected in both SAM P8 and R1 samples. Compared with the correspondingR1 astrocytes, P8 cells had the lower expression of NMDA receptors. By the sole or combined treatments of Aβ （1-42） and tau protein, the expressions of NMDA receptors in P8 astrocytes could be enhanced, particularly by tau or the combined treatment of tau and Aβ. In R1 astrocytes sole Aβ could enhance the expression of NMDA receptors, but tau or the combined treatment of tau and Aβ could lower the expressions. （6） Compared with the corresponding R1 astrocytes, P8 astrocytes in vitro had lower expression of Caspase-3. The sole or combined treatments of Aβ （1-42） and tau could enhance the expressions of Caspase-3, especially by treatment of tau or the combined treatments of tau and Aβ （1-42）. Compared with the expressions in the corresponding R1 astrocytes, there were significantly decreased expression levels of Caspase-3 by treatment of sole tau or by combined treatments of tau and Aβ （1-42）. （7） Compared with the expressions of S100β in the corresponding R1 astrocytes, in vitro P8 astrocytes could express lower levels of S100β without or with the treatments of Aβ（1-42） and/or tau protein. Compared with the expression level in the control group, the expressions of S100β in R1 astrocytes could be significantly enhanced by sole treatment of Aβ（1-42） or tau protein. In P8 astrocytes the expression levels of S100β could be decreased by sole treatment of Aβ（1-42） or tau protein, but can be significantly increased by combined treatments of Aβ（1-42） and tau protein（p<0.05）.（8） The expression levels of GFAP could be significantly increased in both two astrocyte samples with addition of Aβ or tau or mixed solutions. Compared with the expressions in the corresponding controls, the expressions of GFAP could be significantly enhanced by combined treatments of Aβ and tau in both two astrocytes. Compared with the expressions in the corresponding R1 astrocyes, P8 astrocytes had the lower levels of GFAP after treatment of tau protein or combined treatments of tau and Aβ. Conclusions: In the present studies, we first investigated the effects of sole Aβ or tau protein or mixed solutions in two primary astrocyte cultures originated from senescence accelerated mice （SAM P8 and R1）. There were similar but mild different consequences in the expressions of cPKC, HXK, LDH, APP, NMDA receptors, Caspase-3, S100β and GFAP between P8 samples and the corresponding R1 sampels. These data provided clues to study the functional changes of astrocytes during normal aging and aging related disorders.更多还原