本實驗以免疫螢光顯微鏡法以及免疫點墨分析法，探討SR-BI在體外培養大白鼠動脈內皮細胞及平滑肌細胞的分佈及表現。我們將序列495至509的SR-BI氨基酸胜?做為抗原，產生SR-BI免疫抗血清，再以飽和的硫酸銨溶液沈澱法及硝酸纖維膜點墨法進一步純化抗體。大白鼠動脈血管圈培養出初級內皮細胞及平滑肌細胞，以含膽固醇（50μg/ml）的無血清培養液培養0、1、3、24及48小時，之後與抗SR-BI初級抗體及接螢光的抗白兔免疫球蛋白二級抗體反應。結果顯示內皮細胞及平滑肌細胞在培養1到24小時螢光反應隨時間增加而增加，但在 48小時則螢光反應減弱。而免疫點墨分析法之實驗結果顯示，經過含膽固醇的細胞培養液培養後，SR-BI在內皮細胞及平滑肌細胞細胞膜的表現，在培養1到24小時逐漸增加，但在 48小時則降低。我們認為SR-BI在餵食大量膽固醇的細胞之表現可能與促進膽固醇移出細胞有關。 In this study, we have used immunofluorescence microscopy and immunoblotting analysis to investigate the subcellular localization and the expression of SR-BI in the cultured endothelial cells and the smooth muscle cells of rat aorta. A peptide containing residues 495 to 509 from mSR-BI plus an NH2-terminal cysteine was coupled to hemocyanin to generate mSR-BI antiserum in rabbits. The anti-SR-BI antibody was purified by precipitating with ammonium sulfate and followed by nitrocellulose blot method. Cells from primary culture of aortic endothelial cells and smooth muscle cells were incubated with 50μg/ml cholesterol in serum free medium for 0, 1, 3, 24 and 48 hrs before the experiments. The results showed that SR-BI was revealed in the endothelial cells and smooth muscle cells. Immunoblotting analysis also indicated that SR-BI was expressed in the cytoplasmic membranes. The levels of SR-BI expression increased gradually from 1 hr to 24 hrs and decreased at 48 hrs after cholesterol loading. We conclude that SR-BI may facilitate the initial steps of cholesterol efflux in the cholesterol-loaded endothelial cells and smooth muscle cells of rat aorta.
