摘要
目的:探究高糖高脂对成骨细胞增殖和凋亡的影响。方法:以完全培养基为对照组培养基,以含20.0mM葡萄糖(D-glucose)、0.4mM棕榈酸(Palmitic acid,PA)及0.5%牛血清白蛋白(Bovine ser um albumin,BSA)的完全培养基为实验组模拟高糖高脂环境,分别处理成骨细胞0-48h后,显微镜下观察细胞形态,采用CCK-8检测细胞增殖活性,Annex in V/PI双染流式检测细胞凋亡率,Western blotting检测caspase-3及cleaved caspase-3蛋白水平。结果:成骨细胞在高糖高脂环境下培养24h时观察到细胞皱缩变圆,胞间间隙增宽;CCK-8检测提示高糖高脂培养成骨细胞24h时与同时刻对照组相比出现增殖抑制(F_(24h)=24.489,P=0.001),48h后增殖活性(0.027±0.022)较同时刻对照组差异有统计学意义(F_(48h)=272.440,P<0.001);Annex in V/PI双染流式检测显示,24h时高糖高脂组细胞凋亡率为9.68%±3.33%,36h时细胞凋亡率上升至12.89%±3.40%,可达对照组(1.96±1.32)约6.6倍;Western blotting检测发现健康成骨细胞极少表达cleaved caspase-3,高糖高脂可时间依赖性上调cleaved caspase-3蛋白水平,24h时cleaved caspase-3相对蛋白表达量(0.349±0.068)约为对照组(0.146±0.033)2.4倍,36h时(0.468±0.136)达对照组3.2倍(F=9.603,P=0.001)。结论:高糖高脂可时间依赖性抑制成骨细胞增殖,促进凋亡。
Objective：To investigate the effect of the combination of high glucose and free fatty acid on apoptosis and proliferation in osteoblast.Methods：Osteoblasts were treated with high glucose and free fatty acid（D-glucose 20.OmM,PA0.4 mM,BSA 0.5%）for 0-48 h.Phase-contrast microscope was used to observe morphologic alteration of cells.Proliferation was detected by CCK-8.Apoptosis was analysed by Annexin V/PI double dye staining followed by flow cytometry analysis.Western blotting was applied to examine protein expression of caspase-3 and cleaved caspase-3.Results：Combination of high glucose and free fatty acid caused cell shrinkage,rounding as well as detachment after 24 h.CCK-8 data showed that high glucose and free fatty acid inhibited cell proliferation in a time dependent manner compared to control group（F24h=24.489,P=0.001;F48h=272.440,P〈0.001）.Flow cytometry results revealed that in the present of high glucose and free fatty acid,the apoptotic rate was 9.68%±3.33% after 24 h and increased to 12.89%±3.40% after 36 h compared to control group（1.96%± 1.32%）.Western Blotting indicated that healthy osteoblasts rarely expressed cleaved caspase-3.High glucose and free fatty acid increased cleaved caspase-3 protein expression by 2.4 times（0.349± 0.068,F=9.603,P=0.016）and 3.2 times（0.468±0.136,F=9.603,P=0.001）after 24 h and 36 h respectively compared to control group（0.146±0.033）.Conclusion：High glucose and free fatty acid time-dependently inhibit proliferation and increase apoptosis in osteoblast.
作者
吴娟
邱绮虹
杨骥锋
梁敏
WU Juan;QIU Qi-hong;YANG Ji-feng;LIANG Min(Department of Periodontology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China)
出处
《中华老年口腔医学杂志》
2018年第2期65-70,共6页
Chinese Journal of Geriatric Dentistry
基金
国家自然科学基金(项目编号:81170970)
关键词
高糖高脂
成骨细胞
细胞增殖
细胞凋亡
high glucose and free fatty acid
osteoblast
proliferation
apoptosis
