【作者】 隋立森；

【导师】 姜晓丹；

【作者基本信息】 南方医科大学 ， 神经外科学， 2008， 博士

【摘要】 研究背景随着社会的发展,创伤性疾病的发病率呈不断上升的趋势,颅脑损伤的发病率也逐年增高,而有关颅脑损伤后的修复机制尚不完全清楚。大量研究资料显示,对于大多数颅脑损伤患者而言,神经元死亡、神经功能的缺失不可避免。神经元功能丧失后,聚集于大脑内静息状态下的神经干细胞(neural stem cells,NSCs)将被激活。神经干细胞是能自我更新,具有分化成神经元、神经胶质细胞的一类细胞群,可参与修复缺损的神经功能。大量的体内外实验已经证实,成年中枢神经系统(central nerve system,CNS)内有NSCs的存在,这些细胞在一定条件下可以进行增殖、迁移和分化并且发挥一定的功能。但对颅脑损伤后内源性NSCs原位诱导、增殖、分化、迁移及其规律,以及相关调节机制研究尚未完全清楚,研究结果亦各不一致;而以往已证实的Notch信号传导途径在NSCs增殖、分化调控机制中所起的重要作用,是否也调控着颅脑损伤后NSCs的原位诱导、增殖、分化及迁移等,目前尚不清楚。为此,本研究拟通过建立的Feeney’s自由落体实验模型,并通过损伤后大鼠神经行为学评分,从大体及病理学角度,在细胞水平上对损伤模型进行判定;利用免疫组织化学及免疫荧光化学方法研究颅脑损伤后自体NSCs原位增殖、迁移和分化的变化,从而揭示大鼠脑损伤后自体NSCs在颅脑组织修复过程中原位增殖、分化规律;并联合应用免疫学荧光组织化学及蛋白表达检测法观察颅脑损伤后Notch信号通路中Notch1蛋白表达的动态变化,分析该信号蛋白与自体NSCs原位增殖、分化的关系。探讨与NSCs原位增殖分化相关的可能调节机制,为未来临床利用自体NSCs原位诱导、增殖、分化规律促进颅脑损伤后神经功能恢复与重建提供理论依据、奠定重要的实验基础。第一部分大鼠颅脑损伤模型的建立及鉴定目的:通过Feeney’s自由落体实验方法,建立一个便于观察及施加处理因素、控制性好、重复性好,可分级并能反映人类脑损伤特点的颅脑损伤实验动物模型;通过损伤后大鼠神经行为学评分及病理学检测,为进一步研究受损伤脑内的NSCs原位动员规律奠定模型基础。方法:实验采用Feeney’s自由落体硬膜外撞击方法,致大鼠中型颅脑损伤。致伤冲击力为600g.cm;术后按不同的实验分组时间处死动物,于损伤后1～14d连续进行神经行为学观察,测定其神经行为学评分(参照Wayne Clark方法);并从大体及显微镜下观察对照组、假手术组及实验组损伤灶与正常皮层之间的形态差异,以重复测量数据的方差分析统计学方法分析实验结果,判定中型颅脑损伤大鼠模型的成功建立,为下一步颅脑损伤后不同时期的内源性NSCs自身动员、体内增殖分化等研究提供模型保障。结果:实验损伤24小时组同假手术组及对照组的神经行为学评分分别为20.3±3.8、4.8±2.1和2.0±0.8,有显著性差异(F=120.478;p＜0.001);各组之间的组织形态结构有明显差异,实验损伤各组主要表现为低倍镜下见局部组织碎裂、片状出血,损伤灶周围形成明显的水肿带,血管受压变形;高倍镜下见损伤灶周围胶质细胞及神经细胞明显肿胀,细胞核偏移,微血管外间隙扩大,证实了颅脑损伤实验动物模型的成功建立。结论:通过改进的Feeney’s自由落体硬膜外撞击实验,建立了大鼠颅脑损伤模型,并通过神经学行为及组织学变化对模型进行进一步鉴定,提示所建立的大鼠颅脑损伤模型完全达到实验要求,为下一步颅脑损伤后不同时期内源性NSCs自身动员、体内增殖分化规律等研究提供了脑损伤模型依托和保障。第二部分颅脑损伤后神经干细胞原位诱导、增殖分化的实验研究目的:通过建立的颅脑损伤动物模型,以Nestin/NSE、Nestin/GFAP、BrdU/NSE和BrdU/GFAP双标阳性细胞为主要评估指标,利用免疫组织化学及免疫荧光化学方法研究损伤后不同时间点的伤侧脑室下区(Subventricular zone,SVZ)、海马齿状回(Dentate gyrus,DG)区及损伤灶周围的NSCs原位诱导、增殖、分化及迁移的变化规律,为自体原位动员NSCs促进颅脑损伤后神经功能恢复与重建提供可借鉴的理论依据。方法:取对照组、假手术组和实验组不同时间伤侧DG、SVZ区及损伤灶周围的脑组织切片,以巢蛋白(Nestin)和溴脱氧尿苷(Bromodeoxy uridine,BrdU)为NSCs的标记蛋白,采用免疫组织化学方法分别以anti-Nestin、anti-BrdU不同蛋白单抗进行免疫组化染色;以神经元特异性烯醇化酶(Neuron-specific endase,NSE)和胶质原性纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)分别作为神经元和神经胶质细胞的特异标志蛋白,采用免疫荧光化学方法分别进行Nestin/NSE、Nestin/OFAP、BrdU/NSE和BrdU/GFAP双标蛋白抗体免疫荧光染色,以检测脑损伤后、分化自NSCs的神经元和神经胶质细胞;置光镜和激光共聚焦显微镜下观察,计数脑损伤后不同时间点伤侧DG、SVZ区和损伤灶周围皮层的双标阳性细胞数。以重复测量数据和析因的方差分析统计学方法分析实验结果。结果:激光共聚焦显微镜下,各实验组在伤侧DG、SVZ区均出现不同染色强度的Nestin/NSE、Nestin/GFAP、BrdU/NSE和BrdU/GFAP双标抗体细胞染色,Nestin阳性染色主要位于胞质,BrdU主要位于位于胞核。在TBI后的伤侧DG、SVZ、损伤灶周围神经干细胞增殖分裂,均以伤后第3天时最明显,伤侧DG区Nestin、BrdU标记阳性细胞较对照组标记阳性细胞明显升高,分别达到对照组的2～7倍左右,其中以BrdU标记的增殖分裂阳性细胞升高最显著,高于对照组7倍多,提示DG区是神经干细胞增殖分化的重要生发中心,在TBI后,动员了伤侧的DG中神经干细胞,使其增殖分化,并且以增殖分裂细胞成分为主;伤侧SVZ区Nestin、BrdU标记阳性细胞较对照标记阳性细胞明显升高,均达到高于对照组2倍左右,标记阳性细胞数量远多于伤侧SVZ区,其中以Nestin及BrdU标记的代表神经干细胞及增殖分裂阳性细胞升高趋势相同,提示SVZ区是神经干细胞增殖分化的另一生发中心;损伤灶Nestin、BrdU标记阳性细胞较对照组标记阳性细胞明显升高,大部分以增殖细胞标记物BrdU标记阳性细胞为主,且以BrdU/GFAP双标阳性细胞占大部分比例,到伤后第14天时,下降至接近正常水平,统计学上有显著差异(p＜0.001),提示脑损伤后一定时间内,分化自NSCs的胶质细胞可能参与了脑损伤修复过程中的胶质瘢痕形成。结论:本实验条件下,在颅脑损伤后的伤侧DG、SVZ区及损伤灶周围组织Nestin/NSE、Nestin/GFAP、BrdU/NSE和BrdU/GFAP双标阳性细胞均大量表达,均在伤后第3天达到峰值,说明伤侧DG、SVZ区组织中有大量的NSCs增殖分化,增殖分化高峰在伤后第3天。其中,伤后第3天伤侧DG区BrdU/NSE和BrdU/GFAP双标的增殖阳性细胞升高倍数最显著,达对照组7倍多,提示TBI动员了伤侧的DG中神经干细胞,使其增殖分裂,并且以增殖细胞为主;而伤后第3天的伤侧SVZ区Nestin/NSE、Nestin/GFAP、BrdU/NSE和BrdU/GFAP双标阳性细胞的基数明显多于伤侧DG区阳性细胞,伤侧SVZ区神经干细胞及增殖细胞阳性增高趋势一致,提示TBI有效动员了伤侧SVZ中的神经干细胞,使其发生明显的增殖并分化;伤后第3天损伤灶周围BrdU/NSE和BrdU/GFAP双标的增殖阳性细胞升高亦很显著,尤以BrdU/GFAP双标阳性细胞占大部分比例,达对照组6倍多,提示TBI诱导NSCs增殖分化成GFAP标记阳性的胶质细胞为主,并可能参与胶质瘢痕的形成。实验各组到伤后第14天时,Nestin/NSE、Nestin/GFAP、BrdU/NSE和BrdU/GFAP双标阳性细胞数下降至接近正常水平。上述结果提示,DG、SVZ区是NSCs增殖分化的重要的生发中心,且SVZ区是NSCs最重要的生发中心,提示TBI后损伤灶周围以GFAP标记阳性的胶质细胞为主,并与损伤灶周围胶质瘢痕形成密切相关。第三部分大鼠颅脑损伤后神经干细胞原位诱导的Notch信号传导机制研究目的:本实验部分利用免疫学荧光组织化学和蛋白免疫印迹方法测定颅脑损伤后脑室下区及海马的Notch1蛋白基因表达动态变化,并以Notch1/Nestin双标抗体为主要指标,分析Notch1蛋白基因与自体NSCs原位增殖、分化的可能关系。初步探讨与NSCs增殖分化相关的Notch信号通路在NSCs原位诱导中的调控机制,为自体NSCs原位激活治疗颅脑损伤提供实验依据。方法:采用免疫荧光化学及蛋白免疫印迹方法,分别对照组、假手术组及实验各时间点伤侧的DG、SVZ区Notch1/Nestin双标抗体阳性细胞数、Notch1蛋白基因表达动态变化进行定性、定量检测。首先,取实验各组伤侧DG、SVZ区的不同脑组织切片,行Notch1/Nestin免疫荧光双标染色,激光共聚焦显微镜下观察,计数伤侧DG、SVZ区的Notch1/Nestin双标抗体阳性细胞数;然后用Western-blot方法检测实验各组Notch1蛋白基因表达动态变化情况。结果采用析因的方差分析统计学方法分析实验结果。结果:免疫荧光化学结果显示,在TBI后的伤侧DG、SVZ区Notch1/Nestin双标阳性染色细胞,均以伤后第1天时升高最明显,达对照组2～3倍左右;伤后第7天,Notch1/Nestin双标阳性染色细胞仍维持较高水平;到伤后第14天时,下降至接近正常水平,统计学上有显著差异(p＜0.001);Western Blot蛋白免疫印迹方法显示,TBI后的各实验组大鼠伤侧DG、SVZ区组织中Notch1蛋白基因均呈免疫反应阳性,在相对分子量约110KD(Notch1的NICD)处出现阳性条带,在大鼠TBI后第1天时,其海马组织及脑室下区组织Notch1蛋白表达最明显,达峰值,TBI后第7天,Notch1蛋白表达仍维持较高水平。结论:本实验结果提示,TBI后的伤侧DG、SVZ区Notch1/Nestin双标阳性细胞及Notch1蛋白基因表达均在伤后第1天明显上调,达峰值;伤后7天仍维持在较高水平。结合第二部分实验NSCs增殖分化结果,提示TBI后激活Notch信号,在伤后第1天信号活性最强,Notch信号通路激活促进NSCs增殖,在伤后第3天达高峰;伤后第7天Notch信号有所减弱,NSCs增殖减慢;伤后第14天Notch1表达接近正常水平,NSCs增殖接近正常水平。提示TBI激活了Notch信号通路并促进脑内NSCs的原位增殖,参与了脑损伤后NSCs发挥功能修复作用的调控过程。更多还原

【Abstract】 BackgroundWith the development of social industrialization, traumatic brain injury (TBI) is the most common acquired injury in traumatic injury with high morbidity and mortality rates. The molecular mechanisms of restoration in the adult central nervous system (CNS), after TBI. were not clear completely. Recent studies demonstrated the neural function deficit and neuron death are inevitable for most TBI patients. Activation of endogenous neural stem cells (NSCs) under TBI in situ, that exist in the central nervous system of adult mammals, and most of them were at resting stage in vivo in normal. NSCs have the potential of self renewal and can proliferate then differentiate into neurons and astrocytes under some condition, NSCs may be involved in neural function restoration. Recent studies in vivo and in vivto demonstrated NSCs existed in the CNS of adult mammals, and activation of NSCs could further proliferate differentiate and be capable of regenerating function after TBI. The molecular mechanisms underlying increased proliferation differentiation and migration of endogenous NSCs after TBI , still remain unclear and need to be elucidated to aid development of a novel strategy for enhancing neurogenesis after TBI. Although studies demonstrated that activation of a lot of NSCs in the hippocampus dentate gyrus (DG) and subventricular zone (SVZ) could proliferate differentiate into neurons and glial cells after TBI., there are controversial studies on NSCs proliferating and differentiating in the adult CNS after TBI. Notch signaling pathway has been shown to play a fundamental role in regulating the fate of NSCs proliferating and differentiating, however, the complex intrinsic mechanisms of Notch signaling that controls and coordinates NSCs proliferation and differentiation are still poorly understood.In this study, three parts were designed in the experiment. Part one, we developed the brain injury model described by Feeney with some modification, and identified the model on cellar level at the aspect of nerve behavior grade, general and pathology. Part two, we observed the change process of endogenous NSCs proliferating and differentiating in situ by the method immunohistochemistry and immunofluochemistry, found out the rules of endogenous NSCs proliferating and differentiating in the process of self renewal; Part three, we observed the change of Notch1 protein expression in Notch signaling pathway by the means of immunofluochemistry and Proteomics (western-blot), analyzed the relation between Notch1 signal protein expression and NSCs proliferation and differentiation in situ. It is in order to discover the endogenous NSCs proliferating and differentiating in situ by the method immunohistochemistry and immunofluochemistry, elucidate the molecular mechanisms of NSCs proliferation in situ after cerebral injury, and to provide the theorical and experimental foundation on neural function restoration by NSCs proliferating and differentiating.PartⅠObjective: To developing the brain injury model described by Feeney with some modification, that is easy to observe, control, repeat and classify. To identify the model on cellar level from the aspect of nerve behavior grade, general and pathology, etc.Method:The brain contusion device was adopted from the impact method described by Feeney, Moderate brain trauma was made by dropping 20g weight from 30cm. Therefore, the injury wallop is 600g.cm. A series of general and pathology observation were among the different groups. And then, nerve behavior scale was evaluated at 1～14d after TBI, upon Wayne Clark’s standard. The results were compared among the difference groups. These model animals were sacrificed according to the time point of divided groups.Results:Developing the brain injury model by Feeney with some modification, that easy to be observe, controlled, repeated and classify. This experiment showed that there were remarkabl differences among the control, sham and experimental groups on nerve behavior grade, pathology, and so on.Conclusion:A stabilized Moderate brain trauma model was established. The model has practical value in traumatic brain injury experimental and clinical traunatic studies, provides the experimental model foundation for exploring the NSCs in situ activating following TBI. PartⅡObjective:To explore the possible rules of proliferation, differentiation, migration and self-renew of endogenous NSCs in the different regions including SVZ, DG and injury site, at different time points after TBI model was established, by mainly immunohistochemistry and immunofluorescence.Method:At different time points, sections obtained from SVZ, DG and injury site of different experimental groups were stained by anti-Nestin or ant-BrdU antibodies. Double immunofluorescent labeling was performed by using Nestin/ GFAP, Nestin/NSE, BrdU/GFAP or BrdU/NSE antbodies. Immunohistochemical and immunofluorescent staining was photographed with a light microscope and laser confocal microscope. By the help of special software (Leica Q Win), we counted the cell numbers with double labeling in the SVZ, DG and injury site.Results:Under light microscope, Nestin and BrdU immunopositive products with different intensities could be observed in the cells of SVZ and DG in different experimental groups. Nestin-positive staining mainly localized in the cytoplasm, while BrdU immunoreactivity (IR) mainly expressed in the cellular nuclei. The cells numbers with BrdU IR in the experimental group was significant more than the control and sham-operative groups, which was most obvious at 3 days post operation.Under laser confocal microscope, Nestin- and BrdU-positive products with different intensities could be observed in the cells of SVZ and DG in different experimental groups. In the TBI group, the Nestin/GFAP, Nestin/NSE, BrdU/GFAP and BrdU/NSE double labeled positive cells numbers were elevated at 1 day post injury, peaked at 3 days, and then decline at 7 days, recovery to the normal level at 14 days. The changes were most obvious in the SVZ. The glia with abnormal morphologies could be found around the injury site. However, the Nestin-positive cells numbers had no significant changes.ConclusionAccording to the present data, TBI induced in situ the endogenous NSCs that localized in the DG and SVZ proliferation and differentiation. TBI lead to the glia numbers increase abnormally and formed glia scar in the cerebral cortex around the injury cites. Largely Nestin immunoreactive products were observed in the SVZ and DG, which implied many neuronal precursors were mobilized in these regions. Largely BrdU immunoreactive products were observed in the SVZ and DG,, it suggested NSCs began to proliferation and differentiation at 3 days post operation.PartⅢObjective:To observe the change of Notch1 protein expression in Notch signaling pathway by the means of immunofluochemistry and Proteomics (western-blot), analyse the relation between Notch1 signal protein expression and NSCs proliferation and differentiation in situ. And to discover the NSCs proliferating and differentiating in situ by the method of Immunohistochemistry and immunofluochemistry. To elucidate the molecular mechanisms of NSCs proliferation in situ after cerebral jury.Method: The temporal changes of Notch1 (the signal maker of Notch signal pathway) were checked with the ways of qualitative and semi-quantitative investigating, by immunohistochemistry and Western blot respectively. At different time points, sections obtained from SVZ, DG and injury site in different experimental groups were doubly labeled by using Notch1/GFAP and Notch1/NSE antibodies. The cells numbers with double labeling were counted and the OD levels of proteins was detected. Statistics was analyzed by the help of SPSS10.0 software.Results:Under laser confocal microscope, the double labeled cells with Notchl/GFAP、Notch1/NSE at different intensities could be observed in the cells of SVZ and DG in different experimental groups. In the TBI group, the Nestin/GFAP, Nestin/ NSE, BrdU/GFAP and BrdU/NSE double labeled positive cells numbers were elevated at 1 day post injury, remaining high levels at 7 days, and began to decline at 14 days. The immunopostive cells numbers had significant statistics differences between TBI group and control or sham operative groups. Western blot analysis showed that each antibody might specifically recognized the appropriate bands at a molecular weight of approximately 110 KDa (active part of NICD-Notch), and actin control antibody was recognized the bands located at 130 KDa, the Notch content was abundant in the SVZ and DG, remaining high levels a t 7 days, and began to decline at 14 days.Conclusion:Notch1 play a potential regulation role during the course of the in situ activation, differentiation and migration of endogenous NSCs located in the SVZ and DG after TBI.更多还原