探討同半胱胺酸對人類臍靜脈血管內皮細胞細胞 活性及細胞週期之影響

Effects of Homocysteine on Cell Viability and Cell Cycle Progress of Human Umbilical Vein Endothelial Cells

鍾安妮(Annie Zhong)

指導教授 ： 黃莉文

共同指導教授 ： 張基隆(Kee-Lung Chang)

高雄醫學大學/健康科學院/生物醫學檢驗學研究所/碩士(2007年)

摘要

血漿中的同半胱胺酸(Homocysteine; HCy)濃度過高會增加血管內皮細胞自由基的產生，引起內皮細胞的損傷，使得細胞受到氧化傷害而死亡。高濃度的同半胱胺酸已被證實是造成粥狀動脈硬化(Atherosclerosis)的一個獨立危險因子。動脈硬化屬於一種慢性發炎性疾病，且會引起內皮細胞發生功能異常，但其詳細的分子作用機制並不清楚。同半胱胺酸會誘導細胞走向凋亡，且使細胞生長週期停滯，而細胞的生長及死亡與其細胞週期的調控有密切相關。在細胞週期的調控過程中，p21WAF1/CIP1扮演著重要的角色，它是最早被發現且最具廣效性的cyclin-dependent kinase inhibitor (CDKI)。本研究探討藉由加入抗氧化劑Glutathione(GSH)，進而觀察在高濃度同半胱胺酸存在下對血管內皮細胞所造成的效應，及進一步探討同半胱胺酸與細胞週期調控分子p21WAF1/CIP1之間分子作用機制。研究結果顯示，以高濃度同半胱胺酸處理人類臍靜脈血管內皮細胞後，細胞形態由多角形轉變為細長形，且血管內皮細胞的生長會呈抑制現象，甚至死亡。此外，在細胞週期的調控方面，同半胱胺酸可促使細胞週期停滯在G0/G1期，且讓細胞週期調控分子p21WAF1/CIP1之蛋白表現量明顯增加。在觀察單核球黏附血管內皮細胞的情形，同半胱胺酸促使黏附至血管內皮細胞之單核球細胞數目增加，且這種單核球細胞之黏附現象呈現劑量化(dose-dependent)及時間效應關係(time-dependent)。我們也發現，在高濃度同半胱胺酸存在下會誘導血管細胞黏著分子ICAM-1蛋白質的大量表現。若血管內皮細胞先經GSH處理，則實驗發現GSH可以防止同半胱胺酸2mM所引起血管內皮細胞形態的改變，且可提高血管內皮細胞的存活率(cell viability)，並且明顯減少黏附至血管內皮細胞之單核球數目，以及抑制血管內皮細胞內p21WAF1/CIP1, ICAM-1蛋白質的表現量。

由以上研究結果得知，同半胱胺酸可經由增加血管內皮細胞之細胞週期調控分子p21WAF1/CIP1蛋白質的表現，進而抑制細胞週期的進行，最後導致血管內皮細胞的生長停滯甚至死亡；同半胱胺酸亦會促進血管內皮細胞之黏著分子ICAM-1的大量表現，增加血管內皮細胞和白血球之間的接觸並結合，引起更多的白血球進入血管內皮下基質，引起發炎反應，促進粥狀動脈硬化的發生機率。本實驗結果證實，加入抗氧化劑GSH可以部份防止血管內皮細胞免於同半胱胺酸的氧化傷害，進而降低粥狀動脈硬化發生的機率。此結果對心血管疾病的預防與治療學理上，提供重要的學術及臨床應用之參考價值。

關鍵字

同半胱胺酸 ； 細胞週期 ； 人類臍靜脈血管內皮細胞 ； p21waf1/cip1

並列摘要

Hyperhomocysteinemia (HHCy) is an independent risk factor of atherosclerosis. The atherosclerosis can be referred to as a chronic inflammatory disease. In the initiation of atherosclerosis, endothelial cells of the blood vessel loss function firstly. HHCy could cause apoptosis of human umbilical vein endothelial cells (HUVECs) which was proposed by increase of oxidative stress or induced cell cycle arrested. However, the detail molecular mechanisms were not well understood. Cells growth or death is related to cell cycle. The p21Waf1/Cip1, a cyclin-dependent kinase inhibitor, plays important role in controlling cell cycle progress. The present study was designed to examine the effects of homocysteine (HCy) and/or glutathione (GSH) on HUVECs focusing on the cell viability, cell cycle regulation, the relationship with monocyte and cell adhesion molecular expression. We also designed to examine the effects of HCy on the expression of p21Waf1/Cip1 and how the effects were influenced by addition of GSH. Our results showed that higher concentration of HCy inhibited the growth of HUVECs and changed cell morphology from original polygonal shape to elongated shape. HCy also induced cell cycle arrest in G0/G1 phase on HUVECs, and increased the cell cycle progress regulator p21Waf1/Cip1 protein expression. We also found that HCy could increase the monocyte adhesion to HUVECs in time-dependent and dose-dependent way, and HCy could increase the intercellular adhesion molecule-1 (ICAM-1) protein expression. The pre-treatment of HUVECs with GSH could reverse the effects of HCy on the cell viability, the monocyte adhesion to HUVECs, p21Waf1/Cip1 protein and ICAM-1 protein expression.
We clarify that HCy inhibits the cell cycle of HUVECs by increasing the p21Waf1/Cip1 protein expression. Besides, addition of GSH could partially reverse the effects of HCy on endothelial cells. These should be benefit for developing the prevention strategy and treatment for homocysteinemia-induced cardiovascular disease.

並列關鍵字

Homocysteine ； Cell cycle ； HUVEC ； p21waf1/cip1

參考文獻

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