- 學位論文
移植腦衍生神經滋養因子基因轉殖的3T3纖維母細胞對於腦溢血小鼠神經新生的功能恢復影響
Neurogenesis and Functional Recovery after Intracerebral Implantation of Brain-Derived Neurotrophic Factor Transfected 3T3 Fibroblasts in Cerebral Hemorrhage Mice
摘要
在台灣，腦溢血約佔中風患者的四分之一，一個月內死亡率約百分之三十，較腦梗塞高出許多。幸存者亦常存在各式的神經障礙。過去對於中樞神經疾病的治療，我們致力於尋找保護神經的藥物，療效依然難以令人滿意，而腦溢血的治療更是少有進展。手術移除血塊和保守治療對照組間，效果並無統計上差異。強調神經再生的治療模式，成為研究的重點之一。由老鼠的實驗得知，腦室旁及海馬回附近仍存在許多神經幹細胞，於腦部受到損害時有細胞新生的能力。藉由生長因子的給予，可提升內源性神經幹細胞的新生與分化，此種內源性神經細胞新生在許多疾病被認為有治療上的意義。但在腦溢血方面仍然研究仍然缺乏。本實驗的目的即是為了解腦衍生神經滋養因子(BDNF)對於神經細胞新生的助益及腦溢血的治療效果。首先建立綠色螢光蛋白(EGFP)基因轉殖，以及BDNF-EGFP基因轉殖的兩種纖維母細胞穩定細胞株(3T3-EGFP細胞及 3T3-BDNF-EGFP細胞)，經由免疫染色、western blot及ELISA的測定，我們確認3T3-BDNF-EGFP細胞有顯著的BDNF表現。在MTT assay檢測下，確認分泌的BDNF具有生物活性，能幫助神經發育並減少死亡。動物實驗方面，我們將膠原蛋白脢注射入小鼠的紋狀核以模擬人類的基底核出血。以rotarod及核磁共振(MRI)掃描，將篩選後的腦溢血小鼠分為三組：兩組實驗組分別為植入3T3-BDNF-EGFP細胞，以及3T3-EGFP細胞；對照組則接受等量的PBS注射。兩週後犧牲灌流切片，利用EdU, doublecortin, GFAP, NeuN等染色，於顯微鏡下觀察這三組新生的神經細胞數目；藉由行為測試，了解各組的神經功能恢復情形；以及犧牲灌流前的MRI，了解各組腦組織流失的多寡。我們發現兩組接受細胞移植的實驗組，無論在腦室旁或血塊附近，皆有明顯的EdU/GFAP共同標定細胞增加現象；在遷移出去的神經母細胞(neuroblasts)數量方面，則為3T3-BDNF-EGFP組多於3T3-EGFP組，再多於PBS組。我們認為細胞移植能增加神經膠細胞的數量，神經膠細胞會吸引神經母細胞的遷移，BDNF則能避免這些新生細胞的凋亡。因此在MRI檢驗下BDNF治療組有較少的腦組織流失。經由之前的測定，我們的3T3-EGFP細胞亦能分泌少量的BDNF，可解釋為何3T3-EGFP組的遷移性神經母細胞雖然少於3T3-BDNF-EGFP組，但仍多於PBS組。在行為測式方面，我們發現兩組細胞移植組皆比對照組有顯著改善，但3T3-BDNF-EGFP組和3T3-EGFP組並無差異。3T3-BDNF-EGFP治療組雖然有最多的新生神經細胞，但這些細胞大多未成熟，尚未形成新生的神經網路，無法發揮改善功能的效應。經由ELISA的測定，我們發現無論3T3, 3T3-EGFP或3T3-BDNF-EGFP細胞，都能分泌足量的神經生長因子(NGF)及血管內皮生長因子(VEGF)。這可解釋為何兩組細胞移植組有相同的功能進步，我們認為這些進步主要來自於3T3細胞分泌的生長因子的保護作用。基因轉殖的細胞治療，可作為腦溢血神經再生的治療對策之一。
並列摘要
Neurogenesis by activation of the endogenous neural progenitor cells is considered as a potential treatment strategy for brain disease. Brain-derived neurotrophic factor (BDNF), though several articles support its benefit on neurogenesis, there is still some arguments. BDNF has been widely studied in ischemia animals, but rarely in ICH model. In our study, cDNAs of mouse Brain-derived neurotrophic factor (BDNF) were transfected into cell lines of 3T3 fibroblasts. BDNF expression and bioactivity were analyzed by immunocytochemistry, Western blot, Enzyme-Linked Immunosorbent Assay (ELISA), and functional assays. The ICH mouse model was produced by collagenase injection. Hematoma area and brain tissue loss were assessed by magnetic resonance imaging (MRI). Either BDNF transfected 3T3 fibroblasts or 3T3 fibroblasts were implanted as a growth factor source in ICH mice. Neurogenesis and functional recovery was evaluated 15 days post-ICH. BDNF treatment mice have most doublecortin positive cells toward lesions and least brain tissue loss in all groups. Both cell treatment groups have profound newly proliferative glial fibrillary acidic protein (GFAP) positive cells and better functional improvement than the PBS controls. Fibroblast transplantation together with recombinant BDNF treatment has a potential benefit to neurogenesis in ICH mice. The early functional recovery may result from the growth factors that are provided or evoked by the implanted grafts. A potential approach could combine both gene and cell therapy for stroke treatment.
參考文獻
延伸閱讀
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