不同亚型ApoE对创伤后神经元/星型胶质细胞继发性损伤的影响及机制探讨
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摘要
创伤性脑损伤（traumatic brain injury，TBI）是神经外科最常见的疾病之一，被认为是全世界45岁以下人群中最具威胁的致残、致死因素，并且给家庭和社会带来巨大的痛苦和经济损失，已经成为一个全球性的社会经济问题。TBI发生后的损伤包括：原发性损伤和继发性损伤，由于原发性损伤往往无法进行干预，而继发性损伤却因为众多因素参与，具有多个干预靶点，所以继发性损伤过程被认为是影响TBI预后的关键。本课题关注的载脂蛋白E基因（apolipoprotein E gene，APOE）位于19号染色体长臂，编码脑组织中最主要的载脂蛋白——载脂蛋白E（apolipoprotein E，ApoE）。APOE共含4个外显子，主要存在三个等位基因：APOEε2，APOEε3，APOEε4，分别编码三种不同的ApoE，即ApoE2、ApoE3和ApoE4。这三种ApoE分子构成的差别虽然仅限于第112和158位氨基残基不同，但是却导致3种亚型ApoE的生物学功能出现了明显的差异。ApoE4被认为是功能异常的蛋白，常常扮演负性调控因子的角色，而ApoE2和ApoE3的功能则相对正常。作为脑组织最主要的载脂蛋白，ApoE不仅在脂质转运和代谢中发挥作用，还参与神经元和星型胶质细胞的生长和修复、细胞信号传导等过程。我们的前期研究和国内外文献都证实APOE基因多态性能够影响TBI的预后，但是具体机制尚不完全清楚。因此，本课题在前期工作的基础上，以不同亚型ApoE蛋白在TBI后继发性损伤过程中的作用为切入点，通过培养原代神经元和星型胶质细胞并构建机械损伤模型，检测不同亚型ApoE对细胞内钙离子水平（[Ca2+]i）、神经元内线粒体功能以及星型胶质细胞释放谷氨酸的影响，观察不同亚型ApoE对机械损伤后星型胶质细胞活性和修复能力的影响以及与受体ApoER2的亲和力，并探讨不同亚型ApoE在TBI后的继发性损伤过程中发挥的作用及可能方式，以期阐明APOE基因多态性影响TBI预后可能的机制。
一、原代神经元和星型胶质细胞的培养以及机械损伤模型的建立
神经元和星型胶质细胞在脑组织的功能和结构中发挥极其重要的作用，所以对神经元和星型胶质细胞进行体外培养，建立稳定的培养模型，对神经科学研究至关重要；而体外神经元和星型胶质细胞机械损伤模型可控性强，并能在一定程度上模拟TBI后的继发性损伤过程，适合进行机制方面的研究。方法：1.本部分实验提取出生24小时内的APOE基因敲除小鼠（APOE-/-）的脑组织，采取酶消化法获取细胞悬液后，分别进行神经元和星型胶质细胞的培养。其中，神经元的培养基为neurobasal+B27，星型胶质细胞的培养基为DMEM/F12+胎牛血清。2.在进行神经元/星型胶质细胞共培养时，先用传代3次的星型胶质细胞作为滋养层，让其长满培养皿底部，然后将提取的原代细胞直接接种在星型胶质细胞滋养层上，按照2：1的比例加入两种培养基（neurobasal+B27和DMEM/F12+胎牛血清）。细胞培养一段时间后，取状态较好的细胞，分别选用抗GFAP和抗Tubulin Ⅲ的抗体对星型胶质细胞和神经元进行标记，采用免疫荧光技术对细胞进行鉴定。3.采用移液枪枪头作为划伤工具，执移液枪，在显微镜下对培养皿底部的细胞进行划伤，并通过光学显微镜观察细胞划伤情况。结果：1.光镜显示，一种细胞呈“铺路石”样排列，胞体较大呈不规则形，通过免疫荧光检测，发现细胞能被抗GFAP的抗体识别；另一种细胞胞体较小，多呈梭形，有很多较长的突起，且与其它细胞的突起相互交错，形成网络状，通过免疫荧光检测，发现细胞能被抗TubulinⅢ的抗体识别。2.采用免疫双标对细胞进行标记后，发现培养的细胞中部分细胞能被抗GFAP的抗体识别，另一部分细胞能被抗TubulinⅢ的抗体识别，两种细胞相互共存。3.光镜下可见由机械损伤造成的划痕，用移液枪枪头进行机械划伤可控性好。结论：本部分实验，成功进行了神经元和星型胶质细胞的体外培养以及神经元/星型胶质细胞共培养，并且成功构建了细胞的机械损伤模型。
二、不同亚型ApoE对机械损伤后星型胶质细胞释放谷氨酸和神经元线粒体膜电位以及细胞内钙离子的影响及可能机制
[Ca2+]i的变化在TBI后继发性损伤过程中发挥极其重要的作用，而细胞外液中兴奋性氨基酸的浓度以及细胞内线粒体的功能变化均可能与[Ca2+]i改变有关。不同亚型的ApoE则可能通过参与这些过程，不同程度地影响[Ca2+]i。方法：本部分实验将神经元和星型胶质细胞按照加入的ApoE均分为ApoE2组、ApoE3组、ApoE4组和没有加入ApoE的对照组，并进行机械划伤，观察机械损伤后不同时间点各个指标的变化情况：1.收集机械损伤后1h、2h、6h以及未进行机械损伤的星型胶质细胞的细胞外液，通过氨基酸自动分析仪检测各组星型胶质细胞外液中Glu的水平。2.采用荧光探针JC-1对神经元线粒体进行标记，通过流式细胞术检测机械损伤后1h、2h、6h、12h、24h，以及未进行机械损伤的各组神经元内红色和绿色两种荧光的荧光强度比值的变化。3.采用钙离子荧光探针Fluo-3/AM对活细胞内钙离子进行标记，选择488nm的激发波长激发Fluo-3，在激光共聚焦显微镜下观察机械损伤后1h、2h、6h、12h、24h以及未进行机械损伤的各组神经元和星型胶质细胞内钙离子荧光强度的变化。结果：1.在机械损伤2h后，加入不同亚型ApoE的星型胶质细胞外液中Glu水平开始出现差异：ApoE4组的星型胶质细胞外液中Glu水平明显高于ApoE2组和ApoE3组。2.在机械损伤各个时间点，加入不同亚型ApoE的神经元线粒体膜电位变化存在差异，ApoE4组的神经元内线粒体膜电位下降程度明显高于ApoE2组和ApoE3组。3.在机械损伤后各个时间点，ApoE4组的神经元和星型胶质细胞内钙离子荧光强度均明显高于ApoE2组和ApoE3组。结论：机械损伤发生后，不同亚型的ApoE蛋白可导致神经元和星型胶质[Ca2+]i出现不同变化，而这些差异有可能与ApoE对星型胶质细胞释放Glu和神经元线粒体膜电位的影响存在亚型特异性有关，这也可能是APOE基因多态性影响TBI预后的机制之一。
三、不同亚型ApoE对星型胶质细胞修复能力和活性的影响以及与ApoER2的结合情况
星型胶质细胞在脑组织中的作用意义重大，其活性和增殖以及修复能力能够对TBI后的继发性损伤过程产生重要影响。同时，ApoE作为脑组织中最主要的载脂蛋白，能够与LDL受体家族的所有成员，包括LDLR、LRP、VLDLR和ApoER2等进行结合，并通过这些受体发挥作用。方法：本部分实验将神经元和星型胶质细胞按照加入的ApoE蛋白均分为ApoE2组、ApoE3组、ApoE4组和没有加入ApoE蛋白的对照组：1.对各组星型胶质细胞进行机械划伤，并做好标记，在伤后24h、48h、72h进行定点照相，观察各组星型胶质细胞对损伤区域的修复情况，并采用MTT检测各组星型胶质细胞的活性和增殖能力。2.采用免疫双标，分别用抗ApoER2的抗体和抗ApoE的抗体对神经元上的ApoER2和ApoE进行标记，通过激光共聚焦显微镜对二者荧光情况进行合成和检测，判断不同亚型ApoE蛋白与ApoER2结合的情况是否存在差异。结果：1.ApoE4组的星型胶质细胞活性和增殖以及修复能力明显比ApoE2组和ApoE3组差。2.虽然可以观察到ApoE与ApoER2的荧光发生重合，但是不同亚型的ApoE与ApoER2的结合情况却未见明显差异。结论：不同亚型ApoE蛋白与ApoER2结合的情况未见明显差异；但在机械损伤后，加入ApoE4的星型胶质细胞的细胞活性和对损伤区域的修复能力明显比加入ApoE2和ApoE3的星型胶质细胞差。
Traumatic brain injury (TBI) is one of the most common central nervoussystem disorders with high morbidity and mortality, and often bringsdisastrous consequences and heavy economic burden to patients’ familiesand the society.
Apolipoprotein E (ApoE), a34-kDa protein with299aa, is the majorapolipoprotein in the central nervous system. ApoE has three isoforms inhumans: ApoE2, ApoE3and ApoE4, respectively encoded by threedifferent alleles (APOEε2, APOEε3, APOEε4). There are some differencesamong three subtypes of ApoE, and they differ at residues112and158:ApoE3has cysteines (Cys) at112and arginine (Arg) at158, whereasApoE4has arginine at both sites, and ApoE2has cysteines. APOEε4isbelieved to be a negative factor that can result in an unfavorable outcome,and ApoE4, encoded by APOEε4, is considered as a dysfunctional orharmful protein. As the major extracellular lipid carrier in the centralnervous system, ApoE plays a key role in pathological and physiologicalpathways, such as lipid transportation, oxidative stress and inflammatoryresponse.
As shown by many studies, APOE polymorphism can influence theoutcome of TBI, but the exact mechanism remains unclear, and so does theexact mechanism through which ApoE4deteriorates the outcome of TBI. By regulating numerous enzyme systems that affect neuronal physiologyand gene transcription and by influencing the functions of organelles,calcium plays a vital role in regulation of cellular functions. Therefore,variation of intracellular calcium concentration can dramatically influencethe function and metabolism of cells, and sometimes even directly induceapoptosis. Furthermore, intracellular calcium level is believed to play a keyrole in the secondary injury after TBI, so maintenance of intracellularcalcium homeostasis is of critical importance to cells.
Intracellular calcium level may be influenced by many factors after TBI,including increase of extracellular glutamate level, dysfunction of organellesuch as endoplasmic reticulum (ER) and mitochondria, all of which havebeen proved to be related with ApoE. In the present study, we investigatedthe effects of different ApoE on the intracellular calcium level, extracellularglutamate level and mitochondria function in cultured neurons andastrocytes of APOE knock-out mouse after mechanical injury, and studiedeffects of different ApoE on repairing capability of astrocytes, and affinitiesof different ApoE for ApoER2. We also discussed the possible mechanismthrough which APOE polymorphism influence the outcome of TBI.
1. Primary neuron and astrocyte culture and foundation of mechanicalinjury model
Methods: Astrocytes and neurons were obtained from postnatal (24hours)APOE knock-out mouse (APOE-/-) by a standard enzyme treatmentprotocol. The astrocyte and neuron purity were determined by glialfibrillary acidic protein (GFAP) and tubulin Ⅲpositivity respectivelythrough immunofluorescence. Mechanical injury model was produced byscraping adherent cells from a culture dish. All primary astrocytes wereprepared from APOE knock-out (APOE-/-) mouse to avoid the interferenceof ApoE generated by wild-type mouse. Results: Cells can be recognized by GFAP-antibody and tubulin Ⅲ-antibody respectively. The mechanicalinjury area can be observed under microscope. Conclusion: The primaryneuron and astrocyte culture and neuron/astrocyte co-culture weresuccessfully obtained, and mechanical injury model was successfullyestablished, providing foundation for next experiment.
2. Effects of different ApoE on intracellular calcium level, extracellularglutamate level and mitochondria function after mechanical injury
Methods: Three full-length exogenous recombinant human ApoE, i.e.,ApoE2, ApoE3and ApoE4, were obtained from Peprotech (USA). Neuronsand astrocytes were divided into four groups, among which, Group-ApoE4,Group-ApoE3and Group-ApoE2were respectively treated with full-lengthrecombinant human ApoE4, ApoE3and ApoE2, while astrocytes nottreated with ApoE was included as a control group. Neurons and astrocyteswere loaded with Fluo-3/AM, a calcium fluorescent probe, and thefluorescent intensity of cells was individually measured under laserscanning confocal microscope after injury. Mitochondria in neurons waslabeled by JC-1, a fluorescent probe for mitochondria, and the fluorescentintensity was determined by flow cytometry (FCM). Extracellular fluid ofastrocyte was collected, and extracellular glutamate level was analyzed byautomatic amino-acid analyzer. Results: Data of laser scanning confocalmicroscope showed that, the fluorescence intensity of neurons andastrocytes in Group-ApoE4were significantly higher than those inGroup-ApoE2and Group-ApoE3after injury. FCM showed that, thefluorescence intensity of neurons in Group-ApoE4was significantly higherthan that in Group-ApoE2and Group-ApoE3after mechanical injury.Moreover, glutamate level in extracellular fluid of Group-ApoE4wassignificantly higher than that in Group-ApoE2and Group-ApoE3afterinjury. Conclusion: ApoE4may cause higher intracellular calcium level in neurons and astrocytes after mechanical injury, which was associated withextracellular glutamate level and mitochondria function.
3. Possible mechanisms through which different ApoE participate in theprocess after mechanical injury
Methods: Neurons and astrocytes were divided into four groups, amongwhich, Group-ApoE4, Group-ApoE3and Group-ApoE2were respectivelytreated with full-length recombinant human ApoE4, ApoE3and ApoE2,while astrocytes not treated with ApoE was included as a control group.Repairing capability of astrocytes in different groups after mechanicalinjury was measured under microscope. Meanwhile atrocytes survival wasestimated by MTT. Affinities of different ApoE for ApoER2weredetermined by double-immunofluorescent under laser scanning confocalmicroscope. Results: Repairing capability of astrocytes in Group-ApoE4was significantly lower than that in Group-ApoE3and Group-ApoE2, andsurvived astrocytes in Group-ApoE4were significantly less than those inGroup-ApoE3and Group-ApoE2. In addition, no statistically difference offluorescent intensity was found among neurons in different groups.Conclusion: Repairing capability and survival of astrocytes may beimpaired by ApoE4, while different ApoE may have similar affinities whenbinding to ApoER2.
一、原代神经元和星型胶质细胞的培养以及机械损伤模型的建立
神经元和星型胶质细胞在脑组织的功能和结构中发挥极其重要的作用，所以对神经元和星型胶质细胞进行体外培养，建立稳定的培养模型，对神经科学研究至关重要；而体外神经元和星型胶质细胞机械损伤模型可控性强，并能在一定程度上模拟TBI后的继发性损伤过程，适合进行机制方面的研究。方法：1.本部分实验提取出生24小时内的APOE基因敲除小鼠（APOE-/-）的脑组织，采取酶消化法获取细胞悬液后，分别进行神经元和星型胶质细胞的培养。其中，神经元的培养基为neurobasal+B27，星型胶质细胞的培养基为DMEM/F12+胎牛血清。2.在进行神经元/星型胶质细胞共培养时，先用传代3次的星型胶质细胞作为滋养层，让其长满培养皿底部，然后将提取的原代细胞直接接种在星型胶质细胞滋养层上，按照2：1的比例加入两种培养基（neurobasal+B27和DMEM/F12+胎牛血清）。细胞培养一段时间后，取状态较好的细胞，分别选用抗GFAP和抗Tubulin Ⅲ的抗体对星型胶质细胞和神经元进行标记，采用免疫荧光技术对细胞进行鉴定。3.采用移液枪枪头作为划伤工具，执移液枪，在显微镜下对培养皿底部的细胞进行划伤，并通过光学显微镜观察细胞划伤情况。结果：1.光镜显示，一种细胞呈“铺路石”样排列，胞体较大呈不规则形，通过免疫荧光检测，发现细胞能被抗GFAP的抗体识别；另一种细胞胞体较小，多呈梭形，有很多较长的突起，且与其它细胞的突起相互交错，形成网络状，通过免疫荧光检测，发现细胞能被抗TubulinⅢ的抗体识别。2.采用免疫双标对细胞进行标记后，发现培养的细胞中部分细胞能被抗GFAP的抗体识别，另一部分细胞能被抗TubulinⅢ的抗体识别，两种细胞相互共存。3.光镜下可见由机械损伤造成的划痕，用移液枪枪头进行机械划伤可控性好。结论：本部分实验，成功进行了神经元和星型胶质细胞的体外培养以及神经元/星型胶质细胞共培养，并且成功构建了细胞的机械损伤模型。
二、不同亚型ApoE对机械损伤后星型胶质细胞释放谷氨酸和神经元线粒体膜电位以及细胞内钙离子的影响及可能机制
[Ca2+]i的变化在TBI后继发性损伤过程中发挥极其重要的作用，而细胞外液中兴奋性氨基酸的浓度以及细胞内线粒体的功能变化均可能与[Ca2+]i改变有关。不同亚型的ApoE则可能通过参与这些过程，不同程度地影响[Ca2+]i。方法：本部分实验将神经元和星型胶质细胞按照加入的ApoE均分为ApoE2组、ApoE3组、ApoE4组和没有加入ApoE的对照组，并进行机械划伤，观察机械损伤后不同时间点各个指标的变化情况：1.收集机械损伤后1h、2h、6h以及未进行机械损伤的星型胶质细胞的细胞外液，通过氨基酸自动分析仪检测各组星型胶质细胞外液中Glu的水平。2.采用荧光探针JC-1对神经元线粒体进行标记，通过流式细胞术检测机械损伤后1h、2h、6h、12h、24h，以及未进行机械损伤的各组神经元内红色和绿色两种荧光的荧光强度比值的变化。3.采用钙离子荧光探针Fluo-3/AM对活细胞内钙离子进行标记，选择488nm的激发波长激发Fluo-3，在激光共聚焦显微镜下观察机械损伤后1h、2h、6h、12h、24h以及未进行机械损伤的各组神经元和星型胶质细胞内钙离子荧光强度的变化。结果：1.在机械损伤2h后，加入不同亚型ApoE的星型胶质细胞外液中Glu水平开始出现差异：ApoE4组的星型胶质细胞外液中Glu水平明显高于ApoE2组和ApoE3组。2.在机械损伤各个时间点，加入不同亚型ApoE的神经元线粒体膜电位变化存在差异，ApoE4组的神经元内线粒体膜电位下降程度明显高于ApoE2组和ApoE3组。3.在机械损伤后各个时间点，ApoE4组的神经元和星型胶质细胞内钙离子荧光强度均明显高于ApoE2组和ApoE3组。结论：机械损伤发生后，不同亚型的ApoE蛋白可导致神经元和星型胶质[Ca2+]i出现不同变化，而这些差异有可能与ApoE对星型胶质细胞释放Glu和神经元线粒体膜电位的影响存在亚型特异性有关，这也可能是APOE基因多态性影响TBI预后的机制之一。
三、不同亚型ApoE对星型胶质细胞修复能力和活性的影响以及与ApoER2的结合情况
星型胶质细胞在脑组织中的作用意义重大，其活性和增殖以及修复能力能够对TBI后的继发性损伤过程产生重要影响。同时，ApoE作为脑组织中最主要的载脂蛋白，能够与LDL受体家族的所有成员，包括LDLR、LRP、VLDLR和ApoER2等进行结合，并通过这些受体发挥作用。方法：本部分实验将神经元和星型胶质细胞按照加入的ApoE蛋白均分为ApoE2组、ApoE3组、ApoE4组和没有加入ApoE蛋白的对照组：1.对各组星型胶质细胞进行机械划伤，并做好标记，在伤后24h、48h、72h进行定点照相，观察各组星型胶质细胞对损伤区域的修复情况，并采用MTT检测各组星型胶质细胞的活性和增殖能力。2.采用免疫双标，分别用抗ApoER2的抗体和抗ApoE的抗体对神经元上的ApoER2和ApoE进行标记，通过激光共聚焦显微镜对二者荧光情况进行合成和检测，判断不同亚型ApoE蛋白与ApoER2结合的情况是否存在差异。结果：1.ApoE4组的星型胶质细胞活性和增殖以及修复能力明显比ApoE2组和ApoE3组差。2.虽然可以观察到ApoE与ApoER2的荧光发生重合，但是不同亚型的ApoE与ApoER2的结合情况却未见明显差异。结论：不同亚型ApoE蛋白与ApoER2结合的情况未见明显差异；但在机械损伤后，加入ApoE4的星型胶质细胞的细胞活性和对损伤区域的修复能力明显比加入ApoE2和ApoE3的星型胶质细胞差。
Traumatic brain injury (TBI) is one of the most common central nervoussystem disorders with high morbidity and mortality, and often bringsdisastrous consequences and heavy economic burden to patients’ familiesand the society.
Apolipoprotein E (ApoE), a34-kDa protein with299aa, is the majorapolipoprotein in the central nervous system. ApoE has three isoforms inhumans: ApoE2, ApoE3and ApoE4, respectively encoded by threedifferent alleles (APOEε2, APOEε3, APOEε4). There are some differencesamong three subtypes of ApoE, and they differ at residues112and158:ApoE3has cysteines (Cys) at112and arginine (Arg) at158, whereasApoE4has arginine at both sites, and ApoE2has cysteines. APOEε4isbelieved to be a negative factor that can result in an unfavorable outcome,and ApoE4, encoded by APOEε4, is considered as a dysfunctional orharmful protein. As the major extracellular lipid carrier in the centralnervous system, ApoE plays a key role in pathological and physiologicalpathways, such as lipid transportation, oxidative stress and inflammatoryresponse.
As shown by many studies, APOE polymorphism can influence theoutcome of TBI, but the exact mechanism remains unclear, and so does theexact mechanism through which ApoE4deteriorates the outcome of TBI. By regulating numerous enzyme systems that affect neuronal physiologyand gene transcription and by influencing the functions of organelles,calcium plays a vital role in regulation of cellular functions. Therefore,variation of intracellular calcium concentration can dramatically influencethe function and metabolism of cells, and sometimes even directly induceapoptosis. Furthermore, intracellular calcium level is believed to play a keyrole in the secondary injury after TBI, so maintenance of intracellularcalcium homeostasis is of critical importance to cells.
Intracellular calcium level may be influenced by many factors after TBI,including increase of extracellular glutamate level, dysfunction of organellesuch as endoplasmic reticulum (ER) and mitochondria, all of which havebeen proved to be related with ApoE. In the present study, we investigatedthe effects of different ApoE on the intracellular calcium level, extracellularglutamate level and mitochondria function in cultured neurons andastrocytes of APOE knock-out mouse after mechanical injury, and studiedeffects of different ApoE on repairing capability of astrocytes, and affinitiesof different ApoE for ApoER2. We also discussed the possible mechanismthrough which APOE polymorphism influence the outcome of TBI.
1. Primary neuron and astrocyte culture and foundation of mechanicalinjury model
Methods: Astrocytes and neurons were obtained from postnatal (24hours)APOE knock-out mouse (APOE-/-) by a standard enzyme treatmentprotocol. The astrocyte and neuron purity were determined by glialfibrillary acidic protein (GFAP) and tubulin Ⅲpositivity respectivelythrough immunofluorescence. Mechanical injury model was produced byscraping adherent cells from a culture dish. All primary astrocytes wereprepared from APOE knock-out (APOE-/-) mouse to avoid the interferenceof ApoE generated by wild-type mouse. Results: Cells can be recognized by GFAP-antibody and tubulin Ⅲ-antibody respectively. The mechanicalinjury area can be observed under microscope. Conclusion: The primaryneuron and astrocyte culture and neuron/astrocyte co-culture weresuccessfully obtained, and mechanical injury model was successfullyestablished, providing foundation for next experiment.
2. Effects of different ApoE on intracellular calcium level, extracellularglutamate level and mitochondria function after mechanical injury
Methods: Three full-length exogenous recombinant human ApoE, i.e.,ApoE2, ApoE3and ApoE4, were obtained from Peprotech (USA). Neuronsand astrocytes were divided into four groups, among which, Group-ApoE4,Group-ApoE3and Group-ApoE2were respectively treated with full-lengthrecombinant human ApoE4, ApoE3and ApoE2, while astrocytes nottreated with ApoE was included as a control group. Neurons and astrocyteswere loaded with Fluo-3/AM, a calcium fluorescent probe, and thefluorescent intensity of cells was individually measured under laserscanning confocal microscope after injury. Mitochondria in neurons waslabeled by JC-1, a fluorescent probe for mitochondria, and the fluorescentintensity was determined by flow cytometry (FCM). Extracellular fluid ofastrocyte was collected, and extracellular glutamate level was analyzed byautomatic amino-acid analyzer. Results: Data of laser scanning confocalmicroscope showed that, the fluorescence intensity of neurons andastrocytes in Group-ApoE4were significantly higher than those inGroup-ApoE2and Group-ApoE3after injury. FCM showed that, thefluorescence intensity of neurons in Group-ApoE4was significantly higherthan that in Group-ApoE2and Group-ApoE3after mechanical injury.Moreover, glutamate level in extracellular fluid of Group-ApoE4wassignificantly higher than that in Group-ApoE2and Group-ApoE3afterinjury. Conclusion: ApoE4may cause higher intracellular calcium level in neurons and astrocytes after mechanical injury, which was associated withextracellular glutamate level and mitochondria function.
3. Possible mechanisms through which different ApoE participate in theprocess after mechanical injury
Methods: Neurons and astrocytes were divided into four groups, amongwhich, Group-ApoE4, Group-ApoE3and Group-ApoE2were respectivelytreated with full-length recombinant human ApoE4, ApoE3and ApoE2,while astrocytes not treated with ApoE was included as a control group.Repairing capability of astrocytes in different groups after mechanicalinjury was measured under microscope. Meanwhile atrocytes survival wasestimated by MTT. Affinities of different ApoE for ApoER2weredetermined by double-immunofluorescent under laser scanning confocalmicroscope. Results: Repairing capability of astrocytes in Group-ApoE4was significantly lower than that in Group-ApoE3and Group-ApoE2, andsurvived astrocytes in Group-ApoE4were significantly less than those inGroup-ApoE3and Group-ApoE2. In addition, no statistically difference offluorescent intensity was found among neurons in different groups.Conclusion: Repairing capability and survival of astrocytes may beimpaired by ApoE4, while different ApoE may have similar affinities whenbinding to ApoER2.
引文
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