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研究生:潘鐿中
研究生(外文):Yi-Chung Pan
論文名稱:間歇性低氧抑制PC12細胞的增生與分化
論文名稱(外文):Intermittent hypoxia reduces PC12 cell proliferation and differentiation
指導教授:楊昆達楊昆達引用關係
指導教授(外文):Kun-Ta Yang
口試委員:蔡克勵張懷仁楊昆達
口試委員(外文):Ke-Li TsaiHuai-Ren ChangKun-Ta Yang
口試日期:2014-07-18
學位類別:碩士
校院名稱:慈濟大學
系所名稱:生理暨解剖醫學碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:60
中文關鍵詞:間歇性低氧
外文關鍵詞:Intermittent hypoxia
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間歇性低氧在定義上,指的是一種反覆性的缺氧,然後再回復至正常氧分壓的現象。這個現象會出現在臨床疾病上,例如於睡眠呼吸中止症與氣喘等疾病發作時,患者身體都會處於間歇性低氧的狀態。在先前有關於睡眠呼吸中止症的研究中發現,在過程中的缺氧狀態會損害海馬迴的神經細胞,進而造成海馬迴萎縮等傷害,而這些傷害可能會使病患在注意力、短期記憶、及一般智力上產生不可回復性的傷害。在最近的研究中發現,部分的海馬迴區域內含有神經前驅細胞,該細胞可以藉由增生與分化來達到神經再生的作用,所以在本篇實驗中,利用同時具有細胞增生與分化特性的PC12細胞來進行實驗。
在先前的研究中發現,於間歇性低氧的過程中,活性氧物種(ROS)的含量會有上升的現象,並且會影響細胞的增生或產生細胞凋亡,但是其中的詳細機制變化目前都尚未明確。此外,ERK pathway在先前的許多研究中都證明了,該機制在影響細胞增生、凋亡或分化的現象中,都是扮演著重要的角色。ERK pathway的活化會受到一種叫做PP2A的蛋白質磷酸酶調控,所以PP2A也是另一種會影響細胞增生或分化的重要因素之一。
在本篇實驗結果中發現,PC12細胞在經過1–4天的間歇性低氧處理過後,利用流式細胞儀能夠偵測到細胞內的ROS含量有上升的情況,但是這樣的現象並不會使細胞產生細胞凋亡。另外也發現PP2A在mRNA與蛋白質方面也會因為間歇性低氧的處理而有表現量上升的現象,且該變化也同時降低了ERK pathway的活化。這些機制上的變化,影響了PC12細胞的cell cycle ,使其停滯於G0/G1 phase的比例增加,最後使PC12細胞的數量有明顯的下降。而這些機制的變化也同樣影響了NGF所誘導的PC12細胞軸突分化,使細胞分化的比例在經過間歇性低氧處理過後,產生明顯的下降。

Intermittent hypoxia (IH) plays a critical role in sleep breathing disorder-associated hippocampus impairments, including neurocognitive deficits, irreversible memory and learning impairments. IH-induced neuronal injury in the hippocampus may result from reduced precursor cell proliferation and decrease the numbers of postmitotic differentiated neurons. ERK pathway is an important pathway to mediated cell proliferation or cell differentiation, which can be inhibited by protein phosphatase 2A (PP2A). However, the mechanisms underlying IH-induced reactive oxygen species (ROS) generation effects on cell proliferation and neuronal differentiation remain largely unknown.
We used Pheochromocytoma cell (PC12) to find the cell proliferation and differentiation change after IH treatment for 1-4 days. Also to determine the mechanisms change between the ROS, PP2A and ERK pathway. In the results, we found ROS generation significantly increased after 1–4 days of IH without increased cell death, which resulted in increased PP2A mRNA and protein levels. After 3–4 days of IH, ERK1/2 protein phosphorylation decreased, which could be reversed by superoxide dismutase (SOD), 1,10-phenanthroline (Phe), the PP2A phosphorylation inhibitors, okadaic acid (OKA) and cantharidin, and the ERK phosphorylation activator nicotine. In particular, the significantly reduced cell proliferation and increased proportions of cells in the G0/G1 phase after 1–4 days of IH, resulted in decreased numbers of PC12 cells. In addition, the numbers of NGF-induced PC12 cells with neurite outgrowths after 3–4 days of IH were less than those after 4 days of RA, which was also reversed by SOD, Phe, PP2A inhibitors and an ERK activator.
Our results suggest that intermittent hypoxia increased ROS generation and PP2A protein levels. It also decreased ERK pathway activation, which inhibited PC12 cell proliferation and NGF-induced differentiation.
Abstract I
中文摘要 II
第一章 緒論 1
間歇性低氧之簡介 1
阻塞性睡眠呼吸暫停(OSA)之簡介 2
利用間歇性低氧研究阻塞性睡眠呼吸暫停(OSA)之關係 2
間歇性低氧對於神經細胞之傷害和活性氧物種之關係 3
神經再生作用與海馬迴神經之關係 3
活性氧物種抑制細胞的增生與分化之關係 4
ERK pathway與細胞增生或凋亡之關係 5
Protein phosphatase 2A與ROS及ERK pathway之關係 6
PC12細胞運用於在神經與低氧相關的研究 7
實驗研究目的 8
第二章 實驗材料與方法 9
PC12細胞培養 9
間歇性低氧處理 9
粒線體內ROS測量(Mitochondrial ROS measurements) 9
利用流式細胞儀測量細胞死亡(Flow cytometric analysis of cell death) 10
細胞RNA萃取 10
cDNA製作 11
即時定量聚合酶連鎖反應(Real-time PCR) 11
西方墨點法(Western blotting) 12
MTT assay 13
BrdU assay 14
細胞質免疫螢光染色(Immunofluorescent staining) 14
細胞週期分析(Cell cycle analysis) 14
PC12細胞分化比例檢驗 15
第三章 實驗結果 16
間歇性低氧會使PC12細胞粒腺體內的活性氧物種增加 16
間歇性低氧所增加的ROS並不會使PC12細胞產生細胞凋亡 16
間歇性低氧促進ROS的增加會刺激PP2A的表現 17
間歇性低氧所增加PP2A的表現會衰減ERK1/2的活性 18
間歇性低氧造成PC12細胞數量減少 18
間歇性低氧使PC12細胞的細胞週期產生滯留 19
間歇性低氧造成ROS增加與PP2A的活化並抑制下游的ERK1/2的活性,進而抑制細胞增生 19
間歇性低氧造成ROS增加與PP2A的活化並抑制下游的ERK1/2的活性,進而抑制細胞週期 20
間歇性低氧會抑制NGF對PC12細胞的神經分化 21
間歇性低氧會刺激ROS增加與PP2A的活化並抑制下游的ERK1/2的活性,進而抑制NGF刺激的PC12細胞分化 21
第四章 討論 23
實驗結果之總結 23
間歇性低氧對於ROS與細胞凋亡之關係 23
ROS與PP2A之間的相互影響關係 24
ROS影響PP2A與ERK pathway之關係 24
間歇性低氧影響ROS與細胞增生之關係 25
間歇性低氧影響ROS與NGF誘導PC12細胞分化之關係 26
間歇性低氧透過調控ERK pathway來影響PC12細胞的增生與分化 26
第五章 圖表與說明 28
參考文獻 51

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