| 罗 粉,韦 晓,马慧敏,唐 珊,刘 超,祝 群.中性神经酰胺酶在胰岛β细胞脂毒性中的变化及作用[J].现代生物医学进展英文版,2017,17(14):2606-2610. |
| 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 |
| Received:October 09, 2016 Revised:October 28, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.14.002 |
| 中文关键词: 中性神经酰胺酶 棕榈酸 脂毒性 胰岛β细胞 |
| 英文关键词: Neutral Ceramidase Palmitate Lipotoxicity Pancreatic β-cell |
| 基金项目:国家自然科学基金项目（81270898）；江苏省自然科学基金项目（BK20151577） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| ABSTRACT 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 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 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. |
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