呂鋒洲林志立金惠謙Hea Gyum Kim醫學研究所2026-10-299999-12-312016https://handle.ncl.edu.tw/11296/85xzwmhttps://scholars.csmu.edu.tw/handle/123456789/36215阿茲海默症 (Alzheimer’s disease)是老年人口常見的神經性退化性疾病，在老年失智症中所佔比例最高，而隨著全球人口老年化的趨勢，預期失智人口也會隨之快速增加。目前已知阿茲海默症患者腦部有兩種重要的病理特徵，分別是由β類澱粉胜肽 (amyloid β peptide, Aβ)堆積而形成的類澱粉斑塊 (amyloid plaques)以及由Tau蛋白過度磷酸化所組成的神經纖維糾結 (neurofibrillary tangles)，此二病理特徵會阻斷神經彼此溝通和傳遞訊息的功能，造成大量的神經元死亡及引發受影響腦區產生萎縮現象，這些受損的腦區最初主要集中在海馬迴，因此會影響短期記憶。幹細胞 (stem cell)是指原始且未特化的細胞，它們是未充分分化且具有再生各種身體組織器官潛在功能的一種細胞。幹細胞存在所有多細胞組織裡，能經由有絲分裂與分化來分裂成多種的特化細胞，而且可以利用自我更新來提供更多新的幹細胞，所以為了治療被傷害的組織，很多幹細胞相關研究在進行中。近年來新興研究指出透過誘導性多功能幹細胞 (induced pluripotent stem cells, iPSCs)之狀態，其過程為利用導入特定基因或是特定基因產物 (蛋白質)之方式送入體細胞中，使該體細胞變成為具備類似胚胎幹細胞 (Embryonic stem cell, ESCs)般，具有分化成各式細胞之多功能分化能力，並且可以持續增生分裂。而這項新的技術，在2006年首度由日本京都大學山中伸彌教授團隊，將老鼠之纖維細胞製作而成。山中伸彌 (Shinya Yamanaka)因為這項研究與約翰•格登 (John Gurdon)共同獲頒2012年諾貝爾醫學生理獎，山中伸彌教授團隊是將這四個基因Klf4、Oct3/4、Sox2、c-Myc (KOSM) 轉染至體細胞中而得到iPSCs。本研究目的為，將山中伸彌教授團隊使用的四個基因中的致癌因子c-Myc改成Nanog之後，以Klf4、Oct4、Sox2、Nanog (KOSN)這四個基因轉染到SK-N-MC神經細胞裡，稱之為KOSN細胞，使其大量表現Klf4、Oct4、Sox2、Nanog四個基因，活化細胞且製造出類幹細胞之狀態，本研究驗證KOSN細胞是否大量表現Klf4、Oct4、Sox2、Nanog四個基因可以減緩Aβ所造成的神經傷害，且進一步以動物行為實驗證實此類幹細胞之狀態能改善Aβ導致之老鼠神經退化。我們的研究除了評估類幹細胞之狀態是否具幹細胞的能力，並探討是否能對抗Aβ所誘發的神經毒性並進一步達到神經保護的效果。本研究先用Q-PCR及西方墨點法的方法來確認四個基因有成功轉染至神經細胞之後，再使用細胞存活率分析法、免疫螢光染色、免疫細胞化學、西方墨點法等來看有沒有對抗Aβ神經毒性而達到神經保護的效果。其結果發現，具有Klf4、Oct4、Sox2、Nanog這四個基因的KOSN細胞由細胞存活率分析法觀察，在處理Aβ之後KOSN細胞存活量比SK-N-MC細胞明顯較多，因此具有減緩Aβ造成細胞凋亡現象。以免疫螢光染色及西方墨點法也發現KOSN細胞凋亡的比例下降，且活化神經胰島素訊息傳遞中Akt的表現，進而抑制Aβ誘發的pSer307 IRS-1表現，並增加IRS在tyrosine的磷酸化和活化pSer473-Akt/pSer9-GSK3β，進而防止Tau蛋白過磷酸化的現象，最後抑制了Aβ所誘發的氧化傷害，並經由調控Akt路徑來刺激 Nrf2/HO-1的表現；動物實驗中亦驗證了細胞實驗的結果，此外行為試驗中也觀察到，阿茲海默症模擬疾病組不論在學習或是記憶能力上相較正常組大鼠表現較差，但阿茲海默症模擬疾病組移植類幹細胞後的大鼠在學習、記憶及認知上有改善。總體來說，本研究認為類幹細胞能對抗Aβ誘發的細胞凋亡與神經毒性，並抑制阿茲海默症的病理特徵Tau蛋白的過度磷酸化以及達到神經保護的效果。The Alzheimer’s disease (AD) is the most common neurodegenerative disease in the elderly patients. It is a neurodegenerative disease that affects the greatest percentage of people. Also, it is expected to increase rapidly with the global trend of the aging population. There are two important pathological features in the brain of AD patients: one is the amyloid β (Aβ) plaque, and the other is the neurofibrillary tangles (NFTs). These Aβ plaque and neurofibrillary tangle could cause neuronal death and also cerebral atrophy. These damages to the brain are primarily in the hippocampus, which could affect short-term memories in its early stages. Stem cells are unspecialized and undifferentiated biological cells that have the ability to become specialized cells, which can produce more new stem cells. They exist in multicellular organisms and can split into a variety of specialized cells through mitosis and differentiation. Also, they can produce more stem cells by the self-renewal process. Much research is being conducted in order to treat the injured brain tissue. For the use of a transfer specific gene or specific introduced gene product (protein), the iPS cell, which is known as the induced pluripotent stem cell is sent into a somatic cell, so that the somatic cells change like embryonic stem cells (ESCs). Having the ability to differentiate into all kinds of cells, they can also continue the proliferation split. This new technology was pioneered by Shinya Yamanaka’s lab in Kyoto University Japan in 2006. He was awarded the 2012 Nobel Prize, along with Sir John Gurdon, for the discovery of mature cells that can be reprogrammed to become pluripotent. Shinya Yamanaka’s team showed that using four genes transfection, which are Klf4, Oct3 / 4, Sox2, c-Myc could convert adult cells into pluripotent stem cells. In the present study, we used four genes, which are Klf4, Oct4, Sox2, Nanog to make iPS cell-like cells, it is differ from what Yamanaka Shinya research team used, and we then examined whether they protected neuronal cells from Aβ-induced neurotoxicity. We converted c-Myc genes to Nanog genes compare to Yamanaka Shinya research team because c-Myc genes are known to potentially cause the cancer in human body. Furthermore, by animal behavior test we demonstrated whether this iPS cell improve the nerve degeneration from animals'' behavior. Lastly, we also demonstrated whether iPS cells have ability to alternate stem cells, have ability to against neurotoxicity and apoptosis that is induced by Aβ, and also have ability for neuroprotection. First of all, we used Q-PCR and western blotting to prove those four genes (Klf4, Oct4, Sox2, Nanog) are successfully transfected to nerve cells, then we used MTT assays, immunofluorescence, immunocytochemistry, and western blotting to confirm cells that are transfected by those four genes would work against Aβ induced neurotoxicity and could show neuroprotection. From the MTT assays, we observed KOSN cells-which is overexpressed with Klf4, Oct4, Sox2, and Nanog genes-were more survived compare to SK-N-MC cells in vitro. By using immunofluorescence and western blotting, we observed the decreased apoptosis in the KOSN cells, and these KOSN cells activate Akt from insulin signaling pathway so that it could decrease pSer307 IRS-1 expression in the cell treated by Aβ. These KOSN cells also showed results of increased tyrosine IRS-1, and thus, they activate pSer473-Akt and pSer9- GSK3β to prevent hyperphosphorylation of tau protein. From this experiment, we also found that KOSN cells could suppress Aβ induced ROS by stimulating Nrf2/HO-1 by controlling Akt. In addition, our in vivo studies showed that the similar results as in vitro. Our results demonstrated the rats which are classified as having Alzheimer’s disease showed less learning ability and more memory loss compare to normal rats. After we transplanted iPS cells to those rats, the brain function got improved and shows improvement in learning ability and memory. Our results suggested that, iPS cells show ability to against Aβ-induced apoptosis and neurotoxicity, and thus it suppresses hyperphosphorylation of tau protein which is the hall mark of Alzheimer’s disease, and it also shows improved neuroprotection.阿茲海默症β類澱粉胜&#32957誘導性多功能幹細胞胰島素訊息傳遞路徑氧化傷害Alzheimer’s diseaseAmyloid βStem celliPS cellneurofibrillary tanglesthesis