腦源性神經生長因子（BDNF）在腦內出血後促進神經生成之探討

Abstract

幹細胞為具有增生及分化能力的細胞，其中雖然由胚胎所取得的胚胎幹細胞可分化為所有成體的細胞種類，但其來源較少且有倫理的爭議，因此近年來，成體幹細胞相關的研究日顯重要。人類成體擁有神經幹細胞的概念已廣被接受。神經幹細胞主要存在於側腦室下 (subventricular zone; SVZ) 及海馬迴 （hippocampal dentate gyrus)，這些神經幹細胞於成體會持續增生且分化為成熟的神經細胞。 目前已知許多神經相關疾病，如腦中風，腦損傷或神經退化性疾病，皆會對成體神經幹細胞造成刺激或抑制性的影響，然而這些神經疾病和神經再生的關係和意義仍尚未有清楚的定論。腦脊髓液主要由大腦腦室旁的室管膜細胞 (ependymal cells) 所製造，其可由腰椎穿刺法獲得。由於腦組織和腦室間無血液大腦屏障所區隔，因此大腦內的蛋白質有機會經滲透而表現於腦脊髓中。根據過去文獻當大腦出現病變時，腦內所產生的神經營養因子（brain-derived neurotrophic factor，BDNF）可能滲透至SVZ 區而影響了神經幹細胞的增生、移行及分化能力。因此，病患的腦脊髓液可用以研究神經疾病和神經再生的關係。顱內出血會造成嚴重的神經功能受損及致死率，目前臨床上顱內出血的治療以外科手術取出顱內血塊為主，但是效果有限，也缺乏相關神經幹細胞研究，因此本研究探討神經幹細胞於顱內出血的影響。 我們使用膠原蛋白分解酶 (collagenase) 注射入Sprague-Dawley大鼠的腦部形成自發性急性顱內出血的大鼠模式，利用組織化學螢光免疫染色方法觀察7天內該急性出血模式的大鼠SVZ的神經幹細胞的變化。我們取得該出血大鼠及對照組大鼠的腦脊髓液，並加入由大鼠胚胎的腦皮質培養而來的神經幹細胞培養液，利用組織化學螢光免疫染色方法觀察該腦脊髓液對幹細胞增生、移行及分化能力的影響。此外我們亦由罹患自發性顱內出血後併發腦水腫且接受腦室引流手術的病人取得腦脊髓液，以及由正常腦壓水腦症 (normal pressure hydrocephalus) 的患者取得腦脊髓液以作為對照組，當把自發性顱內出血病患及對照組的腦脊髓液加入含有大鼠神經幹細胞的培養液中後，觀察該腦脊髓液對幹細胞的影響，其結果和病患臨床的表現作進一步分析及比較。此外我們利用ELISA法測量腦脊髓液中的BDNF濃度，進一步我們將BDNF抗體加入培養的神經幹細胞，觀察BDNF是否影響神經幹細胞。最後我們藉由腦脊髓液注射入腦出血大鼠，觀察是否影響SVZ的神經幹細胞及治療效果。 在急性出血模式的大鼠雙側SVZ，我們均觀察到在第7天時神經幹細胞有顯著增生，分化的SVZ 神經胚細胞 (neuroblast) 的染色呈現亦有顯著的增加及神經胚細胞移行的增加。另外將大鼠腦出血後第七天的腦脊髓液加入大鼠神經幹細胞培養液中，可發現有顯著的細胞增生及神經胚細胞表現增加。相同的，將病人腦出血後第三天的腦脊髓液加入大鼠神經幹細胞培養液中，亦觀察到有顯著細胞增生，及神經胚細胞呈現的增加。此外我們分別在腦出血大鼠第7天及腦出血病患第3天的腦脊髓液中，測量到有顯著的BDNF濃度增加。進一步若加入BDNF抗體至大鼠神經幹細胞的培養液裡，可觀察到神經幹細胞的增生及神經胚細胞的分化程度均顯著降低。分析腦出血病患的臨床表現相關性，我們發現到出血血腫的容積與BDNF的濃度、神經細胞的增生，以及神經胚細胞的分化，均呈現顯著正相關，而與星狀細胞的分化呈現顯著負相關。在將BDNF注射入腦出血大鼠的腦室實驗中，觀察到大鼠的出血旁區域在第14天時神經幹細胞有顯著增生，發炎及細胞凋亡指標亦有減少的現象。而在BDNF抗體注射入腦出血大鼠的腦室組別則顯著地減少此呈現強度。此外在BDNF組別亦有較好的功能性測試結果。因此我們推論在急性腦出血的影響下，老鼠及病患體內會引起產生內生性BDNF，此內生性BDNF會促進神經幹細胞的增生，分化及移行，並有恢復出血後神經功能的可能。以上結果證明BDNF促進急性腦出血後神經幹細胞活化假說，並支持未來以BDNF治療急性腦出血的可能性。

Stem cells, with their remarkable proliferative and differentiative capabilities, have garnered considerable attention in scientific research. While embryonic stem cells, derived from embryos, can differentiate into any cell type in the adult body, their use raises ethical concerns and faces limitations. In recent years, adult stem cells, particularly neural stem cells, have become a focal point of research due to their potential therapeutic applications. Neural stem cells are crucial players in the regenerative capacity of the brain. They primarily reside in two regions: the subventricular zone (SVZ) and the hippocampal dentate gyrus. In adults, these cells continuously proliferate and differentiate into mature nerve cells, contributing to neural regeneration. Understanding the interaction between neural stem cells and neurological diseases is vital for developing therapy. Numerous neurological disorders, such as stroke, brain injury, and neurodegenerative diseases, can influence the behavior of adult neural stem cells. However, the precise relationship between these conditions and neural regeneration remains unclear. Cerebrospinal fluid, which surrounds the brain and spinal cord, is a potential key player in this relationship. It is known to contain proteins and factors that may affect neural stem cell function. This study explores the impact of cerebrospinal fluid on neural stem cells in the context of acute intracerebral hemorrhage. An experimental model was created by inducing spontaneous acute intracerebral hemorrhage in rats using collagenase. The rats were then observed over seven days to track changes in neural stem cells within the SVZ. The cerebrospinal fluid obtained from both hemorrhagic rats and a control group was introduced to a culture medium containing rat neural stem cells. This allowed researchers to observe the effects on stem cell proliferation, migration, and differentiation. Additionally, cerebrospinal fluid was collected from patients who underwent ventricular drainage surgery after spontaneous intracerebral hemorrhage, along with a control group, to further explore clinical implications. Results from the study demonstrated a significant increase in neural stem cell proliferation and differentiation in the SVZ of rats with acute intracerebral hemorrhage. Similarly, when culturing neural stem cells with cerebrospinal fluid from hemorrhagic rats and patients, there was a notable increase in proliferation and expression of neuroblasts – precursor cells that differentiate into nerve cells. Further analysis revealed a significant increase in the concentration of brain-derived neurotrophic factor (BDNF) in the cerebrospinal fluid of both rats and patients. BDNF is a crucial neurotrophin associated with neuronal survival and growth. Adding BDNF antibodies to the culture medium resulted in a significant reduction in neural stem cell proliferation and differentiation, suggesting a key role for BDNF in the observed effects. Clinical correlations in hemorrhagic patients indicated a positive relationship between hematoma volume, BDNF concentration, neural stem cell proliferation, and neuroblast differentiation. Conversely, there was a negative correlation with astrocyte differentiation, emphasizing the specificity of the observed effects. In our experiment where BDNF was injected into the ventricles of rats with hemorrhage, it was observed that the area surrounding the hemorrhage in the rats showed enhancement of proliferation of neural stem cells by the 14th day. Additionally, indicators of inflammation and apoptosis were reduced. Furthermore, the BDNF group exhibited better functional test results. Therefore, we infer exogenous BDNF also promotes the proliferation, differentiation, and migration of neural stem cells and potentially aids in the recovery of neural function following hemorrhage. In conclusion, this study sheds light on the relationship between cerebrospinal fluid, neural stem cells, and acute intracerebral hemorrhage. The findings suggest that BDNF plays a pivotal role in promoting neural stem cell proliferation, differentiation, and migration in response to hemorrhagic events. Understanding these mechanisms opens the door to potential therapeutic interventions utilizing BDNF for the treatment of acute cerebral hemorrhage. As research progresses, this knowledge may contribute to novel strategies for harnessing the regenerative potential of neural stem cells in the context of neurological disorders.