胰岛素分泌囊泡双层脂膜中胆固醇的可视化与定量研究

摘要

Objective To visualize cholesterol distribution and determine cholesterol mole percentage of insulin secretory granules (SGs) in MIN6 cells. Methods Free cholesterol was labeled with fluorescent cholesterol probe-filipin R in MIN6/phogrin-GFP cells to detect its localization in insulin secretory granules. Furthermore, the subcellular organelles of MIN6 were separated to observe cholesterol distribution by sucrose density gradient centrifugation. Free cholesterol composition in each fraction was measured using thin layer chromatography (TLC). Phospholipids composition was analyzed by ammonium molybdate spectrophotometric method. Results Filipin III-labeled cholesterol localized at cytoplasm and overlapped with insulin secretory granules. Cholesterol accumulated at secretory granules fraction (No. 6 of 8 fractions), with the content of (48.62±3.28)mol%, which was higher than that of organelles at other fractions. Conclusion This is the first study to make visualization and quantification of cholesterol in insulin secretory granules. It is found that insulin secretory granules of MIN6 cells are rich in cholesterol, which suggests that cholesterol plays an important role in insulin secretory granules biogenesis.%目的 通过菲律宾菌素Ⅲ染色和脂质测定法,对胰岛素分泌囊泡双层脂膜中胆固醇的分布进行可视化与定量研究.方法 利用胆固醇荧光探针菲律宾菌素Ⅲ标记稳定表达phogrin-GFP的MIN6细胞,对比细胞内游离胆固醇与胰岛素分泌囊泡的分布情况.通过连续蔗糖密度梯度离心法分离MIN6细胞中的胰岛素分泌囊泡,应用钼酸铵分光光度法和薄层分析法分别测定生物脂膜中磷脂和游离胆固醇含量,从而得到胆固醇所占比例.结果 MIN6细胞内菲律宾菌素Ⅲ标记的游离胆固醇与胰岛素分泌囊泡在胞浆中的分布一致,主要聚集在核周及细胞膜下.此外,连续蔗糖密度梯度离心法分离胰岛素分泌囊泡层中富含胆固醇,该层游离胆固醇摩尔百分比为(48.62±3.28)mol％,高于其余各层的细胞器.结论 本研究首次进行了胰岛素分泌囊泡双层脂膜中胆固醇的可视化与定量测定,发现胰岛素分泌囊泡富含胆固醇,为进一步研究胆固醇对胰岛素分泌囊泡形成的影响提供了理论基础和方法学依据.