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標題: | 蜘蛛網膜下出血對大鼠Subventricular zone 神經再生的影響 Change of neurogenesis capacity in the rat subventricular zone after subarachnoid hemorrhage |
作者: | Wen-Di Lee 李文弟 |
指導教授: | 錢宗良 |
關鍵字: | 蜘蛛網膜下出血,神經再生,SVZ,腦脊髓液,活化小膠質細胞,腦源性神經營養因子(BDNF), SAH,neurogenesis,SVZ,CSF,activated microglia,BDNF, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 蜘蛛網膜下出血是一個具有極高死亡率的疾病。此疾病常會造成病人長期的肢體功能、認知功能或是心智功能的損害。於過去的研究指出當老鼠發生蜘蛛網膜下出血時,其腦部的神經前驅細胞的細胞增生會出現增加的情形。然而目前對於調控蜘蛛網膜下出血之後的神經再生的機制並不是很清楚。因此藉由研究大鼠蜘蛛網膜下出血之後的神經再生情形,不僅能有機會了解此疾病的致病機轉之外,或許亦有利於建立新的治療方式。 本篇實驗我們是藉由抽取0.2毫升的大鼠股動脈血液並將其施打入cisterna magna中來做為蜘蛛網膜下出血的動物模式。老鼠分別於蜘蛛網膜下出血誘發後的第1、3、5和7天犧牲,其腦組織則以免疫組織化學染色之方法進行研究。實驗發現蜘蛛網膜下出血誘發的第7天,於subventricular zone (SVZ)的區域表現Ki67 (細胞進行增生時於細胞核中所表現的一種蛋白) 的細胞出現顯著增加的情形。此外於發病之後的第5和7天,在striatum亦有觀察到表現doublecortin (DCX)的細胞 (特別表現於神經母細胞的一種細胞骨架蛋白)以及Glial fibrillary acidic protein (GFAP)的細胞 (特別表現於星狀細胞的一種蛋白) 皆於數量上呈現最高峰的情形。以上的實驗結果顯示腦部於蜘蛛網膜下出血之後,位於SVZ的神經再生的能力出現了提升的情形。 由於當腦部出現損害之後,於受損區域所生成的生長因子或是細胞激素可能會擴散至SVZ,進一步的進入腦室系統中,因此我們就蒐集了不同發病天數老鼠的腦脊髓液,利用生物體外的方法 (in vitro) 測試其對神經幹細胞的影響。於本篇實驗我們是採集胚胎年齡為15天之大鼠的終腦 (telencephalon) 作為神經幹細胞的來源。首先我們將細胞培養在含有DMEM/F12,N2添加劑和鹼性纖維母細胞生長因子 (bFGF) 的培養液中6天,待其形成初級神經球 (neurosphere) 之後,再以0.5%腦脊髓液進行測試,評估其對神經幹細胞神經再生的影響。於細胞免疫染色的結果中發現,發病第5和7天的腦脊髓液測試組別中,除了表現Ki67的細胞有增加的情形之外,表現DCX或是Tuj-1(特別表現於成熟的神經細胞之細胞骨架蛋白) 的細胞皆呈現增加。此外於酵素結合免疫吸附分析 (ELISA) 中發現於蜘蛛網膜下出血之後,腦脊髓液中的腦源性神經營養因子(brain- derived neurotrophic factor, BDNF)出現顯著的增加。為了進一步的證實BDNF對於促進神經再生以及神經分化的重要性,於是我們分別利用細胞免疫染色以及縮時攝影(time-lapse)的技術進行觀察。結果發現0.5%腦脊髓液的測試組別其促進神經再生以及神經分化的能力相較於陽性對照組(positive control)並無統計上的差異。由於根據先前的研究發現,活化的小膠質細胞(microglia)具有調控SVZ神經再生的能力,因此本篇研究我們想要探討活化的小膠質細胞於蜘蛛網膜下出血的神經再生過程中可能扮演的角色。在ED-1(特別表現於活化的小膠質細胞之蛋白)和Iba-1(表現於小膠質細胞之蛋白)的免疫組織染色的結果發現於發病的第5和7天,在SVZ的小膠質細胞的活化比例出現增加的情形。另外在SVZ的免疫化學染色的結果亦發現BDNF的表現增加,且小膠質細胞和星狀細胞內亦有BDNF的表現。因此由以上的實驗我們可以得知BDNF是扮演促進蜘蛛網膜下出血之後神經再生以及神經分化主要因子。 總結本篇實驗,BDNF是扮演促進蜘蛛網膜下出血之後神經再生以及神經分化主要因子。而BDNF的表現增加則可能與小膠質細胞活化增加或是星狀細胞的調控有關。 Subarachnoid hemorrhage (SAH) is a disease with high mortality rate. Patients suffered from SAH may have long-term physical, neurocognitive, psychiatric, and/or psychological impairments. In rodent brain, increased proliferation of neural precursor cells after SAH has been reported. Nevertheless, the relationship between neurogenesis and SAH has not been fully understood. Investigating the proliferation capacity of neural stem cell (NSC) in SAH rats provides a chance not only to delineate the pathogenesis of this disease, but also to find out the potential application for treatments. In this study, we produced SAH rats by injecting rats with 0.2 ml autologous blood from femoral artery into cisterna magna. Animals were sacrificed on days 1, 3, 5 and 7 after SAH induction and their brains were prepared for the immunohistochemistry study. On day 7 after SAH, an increased amount of the cells positive for Ki67 (a marker for cell proliferation) in the subventricular zone (SVZ) was observed. In addition, the amount of cells positive for doublecortin (DCX, a marker for neuroblast) and glial fibrillary acid protein (GFAP, a marker for astrocyte) in the striatum reached a peak on day 5 and 7 respectively after SAH. These results indicated SVZ neurogenesis was enhanced after SAH. Since the growth factors or cytokines produced around the lesion might diffuse into the SVZ and circulate into the cerebral ventrical system, we collected cerebrospinal fluid (CSF) from different post SAH time points on days 3, 5 and 7 for in vitro study. Furthermore, we isolated the neural stem cells from the embryonic day 15 rat fetal telencephalon. For primary neurospheres, cells were cultured in DMEM/F12 containing N2 supplement and bFGF for 6 days. We then supplemented culture medium with 0.5% traumatic CSF to test its effects on neurogenesis in vitro. We found increased expression of not only ki67 in the neurospheres but DCX and Tuj-1(a marker for mature neuron) in groups day-5 and -7 by immunocytochemistry. Furthermore, ELISA analysis was applied to test the functional factors in the CSF, we found that brain derived neurotrophic factor (BDNF) was significantly increased after SAH. Functional assay including immunocytostaining and timelapse recording was performed to confirm the effect of BDNF on the neurogenesis and neuronal differentiation. We found that both the levels of neurogenesis and neuronal differentiation in the 0.5% CSF treatment group were comparable to the positive control. Additionally, in light of the instructive function of activated microglia in SVZ, we want to explore the roles of activated microglia after SAH. Increased co-staining of ED-1 (a marker for activated microglia)/ Iba-1 (a marker for microglia) was noticed in the SVZ on day 5 and 7 post SAH by the immunohistochemistry study. Furthermore, increased expression of BDNF in microglia and astrocyte was also demonstrated in the SVZ on day 5 and 7 post SAH. Thus, it might suggest that BDNF plays a major role in promoting the neurogenesis and neuronal differentiation after SAH. In summary, increased expression of BDNF which may correlate with the activation of microglial or astrocyte exert an important role in promoting neurogenesis and neuronal differentiation of SVZ after SAH. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53975 |
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顯示於系所單位: | 解剖學暨細胞生物學科所 |
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