脱脂极低密度脂蛋白的制备及其对血管内皮细胞功能的影响

Preparation of degreased very low density lipoprotein and its impact on vascular endothelial cell function

背景极低密度脂蛋白(very low density lipoprotein,VLDL)是动脉粥样硬化的危险因素,由三酰甘油、胆固醇和多种载脂蛋白(apolipoprotein,Apo)构成。由于载脂蛋白分离难度较大,鲜有研究关注VLDL中所含多种载脂蛋白共同作用对动脉粥样硬化的影响。目的使用改良沉淀浓缩法纯化脱脂极低密度脂蛋白(apolipoprotein very low density lipoprotein,ApoVLDL)并评估ApoVLDL对EA.hy926内皮细胞的损伤作用和黏附功能的影响。方法使用沉淀法去除血清中的低密度脂蛋白,然后依次经超滤浓缩离心管浓缩和密度梯度离心分离得到VLDL,最后脱脂得到ApoVLDL,分别使用不同浓度(0μg/mL,50μg/mL,100μg/mL,200μg/mL)的ApoVLDL对EA.hy926内皮细胞进行孵育,以评估ApoVLDL对EA.hy926内皮细胞活力和黏附功能的影响。结果ApoVLDL可以通过改良沉淀浓缩法得到,ApoVLDL粒径为(141.3±0.64)nm,多分散性指数(polydispersity index,PDI)为0.27±0.0076,Zeta电势大小为(-18.73±0.58)mV,颗粒大小均匀,分散系较稳定。使用ApoVLDL孵育EA.hy926内皮细胞24 h后,细胞活力降低明显,呈浓度依赖趋势(P<0.0001)。当ApoVLDL浓度为100μg/mL时,相比对照组,EA.hy926内皮细胞24 h划痕闭合率明显变小(11.10%±0.693%vs 50.57%±2.66%,P<0.001),提示EA.hy926内皮细胞迁移能力受到明显抑制;同时,EA.hy926内皮细胞黏附的白细胞数明显增加(22.13±1.04 vs 7.25±1.58,P<0.01),且EA.hy926内皮细胞VCAM-1 mRNA的表达显著增加(P<0.01)。结论使用改良沉淀浓缩法可以有效制备ApoVLDL。使用ApoVLDL诱导的EA.hy926内皮细胞功能障碍,可为Apo C-Ⅲ诱导内皮功能障碍研究提供一种特异性体外细胞损伤模型。

Background As a major risk factor for atherogenesis,very low-density lipoprotein(VLDL)is composed of triglyceride,cholesterol and multiple apolipoproteins.Due to the difficulty of apolipoprotein isolation,few studies have focused on the effect of multiple apolipoproteins contained in VLDL on atherosclerosis.Objective To purify apolipoprotein very low-density lipoprotein(ApoVLDL)by modified precipitation-concentration method and assess the effect of ApoVLDL on the damage and adhesion function of EA.hy926 endothelial cells.Methods The low density lipoprotein in serum was removed by precipitation method,and then concentrated by ultrafiltration centrifuge tube and separated by density gradient centrifugation to obtain VLDL.ApoVLDL was obtained by VLDL defatting.Different concentrations(blank control group,50μg/mL,100μg/mL,200μG/mL)of ApoVLDL was incubated with EA.hy926 endothelial cells to evaluate the effect of ApoVLDL on the viability and adhesion function of EA.hy926 endothelial cells.Results ApoVLDL could be obtained by improved precipitation concentration method.The particle size of ApoVLDL was(141.3±0.64)nm,the polydispersity index(PDI)was(0.27±0.0076),the Zeta potential was(-18.73±0.58)mV,with uniform size and relatively stable dispersion system.After incubation of EA.hy926 endothelial cells with ApoVLDL for 24 h,the cell viability decreased significantly in a dose-dependent manner(P<0.0001).Cell migration of EA.hy926 endothelial cells was significantly inhibited after treatment with 100μg/mL of ApoVLDL in the scratch motility assay(11.10%±0.693%vs 50.57%±2.66%in the blank control group,P<0.001).At the same time,the number of leukocytes adhered to EA.hy926 endothelial cells increased significantly(22.13±1.04 vs 7.25±1.58 in the blank control group,P<0.01),and the expression of VCAM-1 mRNA in EA.hy926 endothelial cells also increased significantly(P<0.01).Conclusion ApoVLDL can be effectively prepared by improved precipitation concentration method.EA.hy926 endothelial cell dysfunction induced by ApoVLDL can provide a specific cell injury model in vitro for the study of endothelial dysfunction induced by Apolipoprotein C-Ⅲ(Apo C-Ⅲ).