臺灣博碩士論文加值系統

Homocysteine分子具有誘導人類血管平滑肌細胞（vSMC）增生的能力，但其中的機制尚未清楚。在我們這篇論文中，我們發現了Homocysteine誘導人類臍帶動脈平滑肌細胞（HUASMC）表現cyclin D1。當我們以Homocysteine刺激誘導平滑肌細胞在24到48個小時產生增生得現象。並且誘導平滑肌細胞表現cyclin D1蛋白質。
我們再來研究Homocysteine誘導平滑肌細胞調節cyclin D1蛋白質並促使細胞增生其中的機制。Akt與p70S6K訊息路徑被發現到與Homocysteine誘導的cyclin D1表現有關連。Homocysteine會誘導AKT與p70S6K的磷酸化，並且當加入phosphatidylinositol 3-kinase (PI3K) 的抑制劑(LY294002) 與mammalian target of rapamycin (mTOR) 的抑制劑(rapamycin) 同時都可以去抑制Homocysteine誘導的cycin D1蛋白質的表現與細胞增生的現象。我們也發現 integrin的阻抗牲鈦RGD與integrinb1 b3的特異性中和性抗體可以去抑制cyclin D1的表現與AKT以及p70S6K的磷酸化
這個homocysteine誘導的integrin/Akt/p70S6K signaling pathway並且表現cyclin D1蛋白質可能會藉由促進vSMC的增生而去去增快動脈鄒狀硬化損傷的進程

The molecular basis of homocysteine-induced cell proliferation in human vascular smooth muscle cells (vSMCs) remains unclear. We investigated the mechanism by which homocysteine affects the cyclin D1 expression in human umbilical artery smooth muscle cells (HUASMCs). homocysteine treatment for 24 and 48 h induced proliferation of cells. homocysteine increased the protein expression of cyclin D1.
We further examined the mechanism for the regulation of cyclin D1 expression and cell proliferation. Akt and p70S6K signaling pathway was investigated on the responses of cyclin D1 to homocysteine. homocysteine induced the transient phosphorylation of Akt and p70S6K, and treatment with phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin) attenuated the homocysteine-induced up-regulation of cyclin D1 expression and cell proliferation. The integrin antagonist RGD peptide and neutralizing antibodies for b1 and b3 integrin blocked cell proliferation and cyclin D1 expression as well as p70S6K phosphorylation.
This homocysteine-induced integrin/Akt/p70S6K signaling pathway and expression of cyclin D1 may accelerate progression of atherosclerotic lesions by promoting proliferation of vSMCs.

致謝 -------------------------------------------------1
中文摘要 ---------------------------------------------2
Abstract ---------------------------------------------3
第一章 序論 ------------------------------------------4~18
第二章 材料與方法 ------------------------------------19~36
2.1 實驗材料----------------------------------------19~28
A. 細胞株：
B. 抗體 :
C. 實驗儀器 :
D. 實驗藥品、物品 : :
E. Buffer:
2.2 實驗方法----------------------------------------28~36
A. 細胞培養 (Cell culture) :
B. 冷凍細胞：
C. 從臍帶取HUASMC (Human umbilical artery smooth muscle Cells) :
D. RNA反轉錄 (Reverse Transcription ):
E. 聚合酵素鏈鎖反應(polymerase chain reaction, PCR)
F. 定量PCR (Real-Time PCR)
G. PI染色 (Flow PI staining) :
H. 細胞CFSE染色（細胞增生）
I. 收集細胞 (Lysates for western blot) :
J. 蛋白質濃度測定 (Protein assay) :
K. 蛋白質電泳 (SDS-PAGE) :
L. 西方墨點法 (Western blotting) :
第三章 實驗結果 --------------------------------------37~40
第四章 討論 ------------------------------------------41~43
參考文獻 ---------------------------------------------44~48
圖 ---------------------------------------------------49~55

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