中性神经酰胺酶在胰岛β细胞脂毒性中的变化及作用  

The Change and Effect of Neutral Ceramidase in Pancreatic β Cell Lipotoxicity

作　　者：罗粉 韦晓 马慧敏[1] 唐珊[1] 刘超[4] 祝群[1] 

机构地区：[1]南京医科大学第二附属医院内分泌科,江苏南京210011 [2]亳州市人民医院重症医学科,安徽亳州236800 [3]江苏省中西医结合医院内分泌实验室,江苏南京210028 [4]江苏省中西医结合医院内分泌科,江苏南京210028

出　　处：《现代生物医学进展》2017年第14期2606-2610,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81270898);江苏省自然科学基金项目(BK20151577)

摘　　要：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0.5 mM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NCDase siRNA干扰NCDase基因分别建立后,棕榈酸刺激24 h,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24 h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24 h 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p EGFP-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。Objective： To explore the change and effect of NCDase in pancreaticβ 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 cells 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 ceils （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.

关 键 词：中性神经酰胺酶 棕榈酸 脂毒性 胰岛Β细胞 

分 类 号：R-33[医药卫生] R587.1