获得富含胆固醇脂蛋白颗粒的单步密度梯度离心法

Single-step density gradient centrifugation method for obtaining cholesterol-rich lipoprotein particles

摘要:

目的]建立获得富含胆固醇的非高密度脂蛋白(CR-nHDL)颗粒的单步密度梯度离心法,并探究氧化CR-nHDL促泡沫细胞形成的效果。 [方法]采用碘佛醇作为密度介质建立单步密度梯度离心法,从健康志愿者餐后血浆中分离出密度小于1.008 g/mL的CR-nHDL,测定其组分密度,通过酶学法检测胆固醇含量,利用Western blot和蛋白组学分析检测颗粒中的脂蛋白含量。将分离得到的CR-nHDL氧化后与小鼠骨髓来源的巨噬细胞(BMDM)共培养,并与氧化型低密度脂蛋白(ox-LDL)的作用进行比较,通过细胞内脂质蓄积量评估氧化CR-nHDL诱导巨噬细胞泡沫化的能力。 [结果]本法可获得密度小于1.008 g/mL的CR-nHDL。Western blot结果显示,该组分中含有载脂蛋白B100(ApoB100)和载脂蛋白B48(ApoB48),但不含有载脂蛋白AⅠ(ApoAⅠ)。蛋白组学分析结果显示,采用碘佛醇单步法制备的CR-nHDL与经典两步法制备的低密度脂蛋白(LDL)在主要载脂蛋白成分上无显著差异；脂质组学分析结果显示,碘佛醇单步法制备的CR-nHDL中胆固醇酯含量显著高于经典两步法制备的LDL,这提示该方法富集含胆固醇脂蛋白颗粒的效率更高,且由此获得的CR-nHDL经氧化后,相比ox-LDL更能促进巨噬细胞源性泡沫细胞内的脂质蓄积。 [结论]碘佛醇单步密度梯度离心法可快速获取CR-nHDL,其氧化形式具有更强的促巨噬细胞泡沫化能力。

Abstract:

Aim To establish a single-step density gradient centrifugation method for isolating cholesterol-rich non-high density lipoprotein (CR-nHDL) particles and investigate the effect of oxidized CR-nHDL on foam cell formation. Methods A single-step density gradient centrifugation method using ioversol as the density medium was established to isolate CR-nHDL with a density of less than 1.008 g/mL from postprandial plasma of healthy volunteers. The density of the fraction was measured, and the cholesterol content was detected enzymatically. The lipoprotein content in the particles was analyzed by Western blot and proteomics. The isolated CR-nHDL was oxidized and co-cultured with mouse bone marrow-derived macrophages (BMDM), and its effect was compared with that of oxidized low density lipoprotein (ox-LDL). The ability of oxidized CR-nHDL to induce macrophage foam cell formation was assessed by measuring intracellular lipid accumulation. Results This method successfully isolated CR-nHDL with a density of less than 1.008 g/mL. Western blot results showed that this fraction contained apolipoprotein B100 (ApoB100) and apolipoprotein B48 (ApoB48) but not apolipoprotein AⅠ (ApoAⅠ). The results of proteomic analysis showed that there was no significant difference in the major apolipoprotein components between CR-nHDL prepared by ioversol method and low density lipoprotein (LDL) prepared by classical two-step method; lipidomics analysis results showed that the cholesterol ester content in the CR-nHDL prepared by the ioversol method was significantly higher than that in the LDL prepared by the classical method, which suggested that this method was more efficient in enriching cholesterol-containing lipoprotein particles, and the oxidized CR-nHDL obtained from this method could promote lipid accumulation in macrophage derived foam cells more than ox-LDL. Conclusion The single-step density gradient centrifugation of ioversol can quickly isolate CR-nHDL, and its oxidized form has a stronger ability to promote macrophage foam cell formation.