高密度脂蛋白可藉由膽固醇逆運送的方式來防止動脈硬化的形成，然而對高密度脂蛋白如何將周邊細胞多餘膽固醇攜出的機制尚不清楚。Caveolae是細胞表面上稱為“膜小囊”的凹陷構造，被認為是細胞內膽固醇釋出的出口。而Caveolin-1 是caveolae的主要結構蛋白，推測和細胞內的膽固醇運送有關，但caveolin-1 與caveolae 在膽固醇釋出中扮演的角色還有很多爭議。最近有報導指出與膽固醇釋出有關的另一種膜蛋白“ABCA1”蛋白，具有將細胞膜上膽固醇轉移至apoA-1 的能力。因此，對於cavoelin-1 與ABCA1 在高密度脂蛋白移除細胞膽固醇的過程中所扮演的角色及相互關係有待進一步確認。本實驗室最近利用免疫螢光染色發現在含有高膽固醇的內皮細胞中，caveolin-1、ABCA1及高密度脂蛋白有共同分佈的現象。利用化學聯結法、免疫沉澱法證實HDL 與ABCA1直接連結，ABCA1 與caveolin-1 直接連結，但caveolin-1 卻與高密度脂蛋白未直接連結。故我們推測ABCA1 極可能位於caveolae 上，擔任由高密度脂蛋白所調控的膽固醇外送時中間媒介的角色。為了更進一步探討ABCA1 及caveolin-1 在結構功能上的關係，本計畫將以基因轉殖及RNAi技術分別增加及降低內皮細胞內caveolin-1 的表現，探討caveolae 的結構消長及對ABCA1 的分佈、表現及膽固醇外送的影響。 It has been recognized that the high-density lipoprotein (HDL) plays an important role in the prevention of atherogenic plaque formation. However, there is still considerable debate about the mechanisms by which HDL removes excess cholesterol from peripheral cells. Caveolin-1, the main structural protein of caveolae is involved in the transport of excess cholesterol to HDL at the cell surface. However, the roles of caveolin-1 in cholesterol efflux is still controversial. The ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein , is also reported to mediate the efflux of excess cellular cholesterol to apoA1 and HDL. However, the interactions among ABCA1, caveolin-1 and HDL are not fully understood. In our previous studies, immunofluorescence confocal microscopy showed that the HDL appeared to colocalize with ABCA1 and caveolin-1 on the cell surface. The chemical cross-linking and immunoprecipitation analysis revealed that ABCA1 binds directly to both HDL and caveolin-1, whereas HDL does not bind directly to caveolin-1. These studies provide evidence for a direct interaction between HDL and ABCA1, ABCA1 and caveolin-1, but not HDL and caveolin-1, indicating that ABCA1 may act as an intermediate substrate between HDL and caveolin-1 on the cell surface during cellular cholesterol efflux. In order to investigate the functional relationships between ABCA1 and caveolin-1 on the HDL-mediated cholesterol efflux, the caveolin-1 will be either over-expressed by transfection of pcDNA-CAV vector or down-regulated by 3 RNA interference in the cholesterol-loaded endothelial cells. The distribution and expression of caveolin-1 and ABCA1 in the cells will be evaluated by western blot, confocal and electron microscopy. The cholesterol efflux will be determined.
