臺灣博碩士論文加值系統

低密度脂蛋白(low density lipoprotein; LDL)容易被氧化形成氧化態低密度脂蛋白(oxidized low density lipoprotein; oxLDL)。oxLDL會造成組織和細胞的損傷。Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1)是一種細胞膜蛋白，且為內皮細胞最主要的oxLDL受體，負責將oxLDL胞飲(endocytosis)入細胞，並對細胞產生毒性。腦血管內皮細胞(cerebral endothelial cells; CECs)為血腦障壁(blood brain barrier; BBB)主要組成之一。在CECs中，LOX-1基因的表現量會被誘導，但是其中調控的機制卻還不清楚，因此本研究主要探討oxLDL誘導CECs中LOX-1基因表現之訊息傳遞路徑。
本實驗室先前的研究結果顯示，oxLDL會導致CECs凋亡，因此我們首先探討LOX-1是否在此凋亡反應中扮演重要的角色。利用LOX-1 siRNA達到knockdown的效果，並以顯微鏡觀察細胞形態和利用流式細胞儀評估凋亡細胞所佔的比例，發現LOX-1 siRNA有效的抑制了oxLDL所導致的細胞凋亡反應，顯示了LOX-1可能參與調控此凋亡反應。接續以RT-PCR和real-time PCR分析腦血管內皮細胞中LOX-1 mRNA，發現oxLDL的確會誘導LOX-1基因表現，並且有時間與濃度效應。
在訊息傳遞路徑探討上，於先前研究中發現oxLDL會造成CECs中活性氧分子(reactive oxygen species; ROS)上升，而NF-κB 為對氧化還原反應靈敏度高的訊息傳遞轉錄因子(redox sensitive transcription factor)，因此接續利用核質分離的方式分析氧化壓力相關的轉錄因子NF-κB之轉位(translocation)現象，發現CECs經oxLDL處理後，NF-κB轉位入核的量有時間效應的增加，並在3小時達最大轉位量，進ㄧ步以NF-κB 的抑制劑Bay 11-7085進行前處理後，oxLDL誘導LOX-1 mRNA表現的情形會受到抑制，實驗結果顯示oxLDL的確會透過活化NF-κB 誘導LOX-1基因表現。
接著依序往NF-κB 訊息傳遞路徑上游探討，以免疫轉漬法測定oxLDL對CECs中protein kinases的磷酸化情形，結果顯示CECs經oxLDL處理後，IKK磷酸化會在30分鐘時增加，ERK1/2磷酸化的增加出現於15分鐘，而MEK1/2磷酸化增加出現在5分鐘，Raf的磷酸化則在1分鐘後開始增加，以免疫沉澱的方式也可看到活化態Ras的增加。再進ㄧ步利用LOX-1 siRNA方式探討LOX-1受體在oxLDL誘導LOX-1基因表現之訊息傳遞路徑中所扮演的角色，實驗證實LOX-1 siRNA有效的抑制了oxLDL對LOX-1基因的誘導，同樣的oxLDL所引發的NF-κB轉位現象以及Raf磷酸化也會受到抑制。
研究結果顯示，oxLDL的確會透過LOX-1受體導致CECs產生細胞凋亡反應，並經由LOX-1受體活化細胞內Ras/Raf/MEK/ERK的訊息傳遞路徑，進而磷酸化蛋白激酶IKK導致轉錄因子NF-κB 的轉位入核的量增加，使得LOX-1基因表現量的增加。

Low density lipoprotein (LDL) can be oxidized to oxidized LDL (oxLDL). OxLDL can damage a variety of tissues and cells. Lectin-like oxLDL receptor-1 (LOX-1) is a membrane protein specifically mediating endocytosis of oxLDL and its toxicity to cells. Cerebral endothelial cells (CECs) are important components in blood brain barrier (BBB). LOX-1 is inducible in CECs, but its regulatory mechanism is still unknown. This study is aimed to evaluate the signal-tranducing mechanism of oxLDL-induced LOX-1 gene expression using mouse CECs as the experimental model.
Previous studies in our lab revealed that oxLDL could cause apoptosis in CECs. Thus, one of the specific aims in this study was to determine if LOX-1 receptor had a critical role in oxLDL-induced apoptosis of CECs. Administration of CECs with oxLDL increased LOX-1 mRNA production in concentration- and time-dependent manners. In the LOX-1 siRNA experiment, we detect the distribution of apoptotic cells using microscope and flow cytometry. The data show that LOX-1 siRNA effectively inhibited oxLDL-induced apoptosis.
Thus, the other aim of this study was to evaluate the signal-tranducing mechanism of oxLDL on LOX-1 gene expression. Previous studies in our lab revealed that oxLDL could increase reactive oxygen species (ROS) in CECs. NF-κB is a redox sensitive transcription factor. In parallel with LOX-1 mRNA induction, oxLDL activated transcription factor NF-κB and promoted its translocation from cytoplasm into nuclei. Administration of Bay 11-7085, an NF-κB inhibitor, then consequently inhibited LOX-1 mRNA production.
NF-κB activated by upstream protein kinases. IKK, an upstream kinase for NF-κB activation, was activated after exposure to oxLDL for 30 min. ERK1/2, an upstream kinase for IKK activation, was activated after exposure to oxLDL for 15 min. When exposed to oxLDL for 5 min, MEK1/2, a kinase for ERK1/2 activation, was activated. Besides , Raf, a kinase for MEK 1/2 activation, was activated by oxLDL. Ras, a kinase for Raf, was also activated by oxLDL. We also observed that oxLDL-induced LOX-1 gene expression, NF-κB translocation and Raf phosphorylation were inhibited in the LOX-1 siRNA experiment.
This study has shown that LOX-1 mediated oxLDL-induced apoptosis of CECs, and oxLDL induced LOX-1 gene expression via a Ras/Raf/MEK/ERK-dependent activation of NF-κB.

目錄 I
縮寫表 IV
表目次 V
圖目次 VI
中文摘要 VIII
英文摘要 X
第一章 緒言 1
第一節 氧化態低密度脂蛋白 1
第二節 氧化態低密度脂蛋白受體 3
第三節 氧化態低密度脂蛋白影響腦血管內皮細胞 6
第四節 血腦障壁與腦血管疾病之關聯性 7
第五節 氧化態低密度脂蛋白、活性氧分子及NF-κB轉錄因子
之關連性 8
第六節 活性氧分子、Ras/Raf/MEK/ERK cascade及NF-κB之關聯性 10
第七節 研究假說及研究目標 11
第八節 實驗設計及說明 13
第二章 材料與方法 17
第一節 實驗材料 17
第二節 氧化態低密度脂蛋白之製備 18
第三節 小鼠大腦血管內皮細胞之製備 20
第四節 細胞存活率測定 21
第五節 RNA萃取 22
第六節 反轉錄聚合酶連鎖反應分析 23
第七節 即時聚合酶連鎖反應分析 24
第八節 細胞質蛋白及細胞核蛋白分離 25
第九節 免疫轉漬法 26
第十節 Ras活性分析 28
第十一節 核醣核酸干擾術 30
第十二節 基因轉染 31
第十三節 統計分析 32
第三章 結果 33
第一節 LOX-1受體在oxLDL導致CECs細胞凋亡中所扮演的角色
33
第二節 oxLDL對CECs中LOX-1基因表現的影響 35
第三節 oxLDL會透過活化NF-κB誘導LOX-1基因表現 37
第四節 oxLDL對CECs中protein kinases之影響 38
第五節 LOX-1受體在oxLDL誘導LOX-1基因表現之訊息傳遞路徑中所扮演的角色 40
第四章 討論 43
第一節 LOX-1受體在oxLDL導致CECs細胞凋亡中所扮演的角色
43
第二節 oxLDL對CECs中LOX-1基因表現的影響 44
第三節 oxLDL會透過活化NF-κB誘導LOX-1基因表現 45
第四節 oxLDL對CECs中protein kinases之影響 46
第五節 LOX-1受體在oxLDL誘導LOX-1基因表現之訊息傳遞路徑中所扮演的角色 49
第六節 結論 50
第五章 參考文獻 51
第六章 圖及表 63

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