神經膠質瘤是很常見的腦部腫瘤，世界衛生組織將之分為四個等級，良性的神經膠質瘤被分在第一級 ( Pilocytic astrocytomas) 及第二級 ( Astrocytomas)，而惡性的神經膠質瘤則被分為第三級(Anaplastic Astrocytomas, AA) 及第四級 (Glioblastoma Multiforme, GBM)，雖然第三級及第四級都是惡性腫瘤，但是第三級的二年存活率約為50 %而第四級的則不到20 %。腦瘤的形成機制仍然不是很清楚。
本研究主要探討p27 kip1蛋白在人類惡性腦瘤形成中所扮演之角色，分別以核醣核酸保護分析法 (RPA)、西方墨點法及免疫組織染色來分析腦瘤檢體，檢體一共有59個，其中正常腦組織有5個，astrocytomas有30個而GBM有17個，以RPA偵測正常腦組織及腦瘤的mRNA發現p130、p107、Rb及p27 kip1絕大多數有表現，值得注意的是p27kip1的mRNA在astrocytomas和GBM檢體中全部都有表現，而且在腦瘤表現量都比正常組織高。
PI3K/Akt訊號途徑調節許多細胞功能，例如存活及細胞增生，活化的Akt會將p27kip1磷酸化，這可能會使細胞週期由G1期移動到S期，而磷酸化的p27 kip1會被Skp2辨識而被分解，我們以西方墨點法發現活化的Akt及p27 kip1在astrocytomas及GBM都有過量表現。活化的Akt (p-Akt)在正常腦組織之表現比例為100 %，astrocytomas為90 %而GBM為82 %，不活化Akt則是全部都有表現，至於Skp2的表現在正常腦組織比例為20 %、astrocytomas為87 %而GBM則有為71 %。此外在免疫組織染色方面發現GBM在p27 kip1與p-Akt之間有顯著相關 (P = 0.0236)。所以腦瘤的p27在mRNA有過量表現，而其蛋白也有表現，可能是受到p-Akt及Skp2的影響。

Gliomas are the most common human brain tumors and are divided into four stages by WHO classification scheme. Benign gliomas are defined as grades I (Pilocytic astrocytomas) and II (Astrocytomas), whereas grade III (Anaplastic Astrocytomas, AA) and grade IV (Glioblastoma Multiforme, GBM) are malignant. Although both grades III and IV are malignant, the prognoses for these tumors are quite different. The 2-year survival rate for grade III gliomas is 50%, and grade IV is < 20 %. Mechanisms of tumorigenesis are not exactly elucidated in brain tumor cells.
The thesis is to study the role of p27 kip1 in human malignant brain tumors. The experimental methods include ribonuclease protection assay (RPA), western blotting, immunohistochemical staining and immunocytochemical staining. mRNAs of p130, p107, Rb, p53 and p27 kip1 in normal brain tissues and brain tumors were overexpressed in most case. The p27kip1 mRNA were expressed in all astrocytomas and GBM, and mRNA quantity of p27kip1 were more in brain tumors than in normal brain tissues.
PI3K/Akt pathway regulates several cellular functions such as cell survival and cell proliferation. Active Akt can phosphorylate p27kip1 that may contribute cell cycle from G1 phase to S phase. Skp2 identifies phospho-p27kip1 and promotes p27kip1 degradation. We found p27kip1 overexpression and Akt activation in astrocytomas and GBM. The expression of p-Akt were found in 20 %, 87 % and 71 % in normal brain tissues, astrocytomas and GBM, respectively. Expression of p27kip1 and p-Akt has shown significant correlation in GBM (P = 0.0236). Overexpression of p27kip1 mRNA in brain tumors may be consequence of p-Akt and Skp2.

縮寫----------------------------------------------------------------------p.1
中文摘要----------------------------------------------------------------p.2
英文摘要----------------------------------------------------------------p.4
前言----------------------------------------------------------------------p.6
細胞週期--------------------------------------------------------------p.6
細胞週期條調節基因p27kip1--------------------------------------p.7
PI3K/Akt訊號途徑--------------------------------------------------p.8
腦瘤的分類------------------------------------------------------------p.11
實驗材料與方法---------------------------------------------------------p14
實驗結果------------------------------------------------------------------p.23
腦瘤的細胞週期相關基因mRNA表現--------------------------p.23
p27kip1、 p-Akt與Skp2蛋白質的表現--------------------------p.24
以LY294002處理細胞----------------------------------------------p.25
LY294002抑制Akt活性對p27kip1的影響----------------------p.25
U0126抑制Erk1/Erk2活性對p27kip1的影響------------------p.26
討論------------------------------------------------------------------------p.28
參考資料------------------------------------------------------------------p32
附圖------------------------------------------------------------------------p.38

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