中性神经酰胺酶在胰岛β细胞脂毒性中的变化及作用

摘要

Objective:To explore the change and effect of NCDase in pancreatic β cell lipotoxicity.Methods:Cell viability was detected by MTT assay after being incubated with 0.5 mM palmitate for different time periods and cell proliferation was measured by MTT assay after transfected with pEGFP-C3-NCDase or NCDase siRNA.NCDase activity and protein expression were investigated by HPLC and Western Blot assay,respectively,after INS-1 ceils were treated with 0.5 mM palmitate for 24 h.Stable clones of INS-1 cell line transfected with recombinant plasmids pEGFP-C3-NCDase and pEGFP-C3 vector were established and NCDase siRNA was transiently transfected into INS-1 cells.Then they were incubated with palmitate for 24 h.The apoptosis rate was detected by flow cytometry.Results:Compared with the controls,cell viability was decreased by (52± 3.2) ％ (P＜0.01),treated with 0.5 mM palmitate for 24 h.Compared with BSA group,NCDase activity and protein expression were markedly inhibited (P＜0.01).NCDase overexpression markedly enhanced-cell proliferation,while NCDase siRNA significantly inhibited-cell proliferation,compared with BSA group (both P＜0.05).Compared with pEGFP-C3 plus palmitate group,NCDase overexpression alleviated palmitate-induced apoptosis in INS-1 cells (P＜0.01).In contrast,NCDase siRNA significantly enhanced palmitate-induced apoptosis (P＜0.01),compared with con.siRNA plus palmitate group.Conclusion:NCDase activity and protein expression were inhibited by palmitate.In addition,NCDase overexpression promoted cell proliferation and protected against palmitate-induced lipotoxicity in pancreatic β-cell.%目的:探讨中性神经酰胺酶(NCDase)在胰岛β细胞脂毒性中的变化及作用.方法:采用0.5mM棕榈酸作用INS-1细胞不同时间,用MTT法检测细胞活力;细胞内分别建立NCDase基因过表达和干扰后,用MTT法检测细胞的增殖活力;棕榈酸刺激细胞24 h,HPLC法和Western Blot法检测NCDase活性和蛋白表达;重组质粒pEGFP-C3-NCDase过表达NCDase基因和NCDasesiRNA干扰NCDase基因分别建立后,棕榈酸刺激24h,用流式细胞术检测细胞凋亡.结果:与对照组相比,棕榈酸刺激24h时细胞抑制率显著降低(52±3.2)％(P＜0.01);与BSA对照相比,棕榈酸刺激24h NCDase活性显著被抑制(P＜0.01),NCDase蛋白水平也被显著下调(P＜0.001);与BSA对照组相比,过表达NCDase显著促进细胞的增殖,然而NCDase干扰显著抑制细胞的增殖(P＜0.05);与pEGFP-C3+棕榈酸组相比,pEGFP-C3-NCDase显著缓解棕榈酸诱导的细胞凋亡(P＜0.01);与con.siRNA+棕榈酸组相比,NCDase siRNA显著促进了棕榈酸诱导的细胞凋亡(P＜0.01).结论:棕榈酸刺激后抑制β细胞NCDase活性和蛋白表达,NCDase过表达促进β细胞增殖并且在β细胞脂毒性中起着保护作用.