油酸诱导的HepG2肝细胞脂性凋亡模型的建立
首发时间:2019-03-20
摘要:目的探讨油酸(Oleic acid, OA)对人肝癌细胞(hepatocellular carcinoma cells, HepG2)脂质代谢的影响,建立HepG2细胞脂毒性损伤模型。方法不同浓度(0、0.1、0.3、0.5、0.7、0.9 mmol/L)OA刺激HepG2细胞24 h,用cck-8比色法检测细胞增殖情况,LDH释放法检测细胞毒性,油红O染色法定性观察细胞脂质沉积情况,并测定细胞内甘油三酯(TG)、丙二醛(MDA)、超氧化物歧化酶(SOD)以及培养液中谷丙转氨酶(ALT)和谷草转氨酶(AST)水平以确定最佳模型浓度;用细胞周期检测试剂盒检测细胞周期情况,Hoechst 33342染色和Annexin-V FITC-PI 流式细胞术检测细胞凋亡情况。结果以0.7 mmol/L OA刺激HepG2细胞24 h时,细胞增殖水平出现一定程度的抑制(p < 0.05),LDH细胞毒性处于较低水平,脂质沉积显著 (p < 0.05),ALT,AST释放量、MDA含量显著增加(p < 0.05),SOD活性明显下降(p < 0.05),细胞周期被抑制在G1/S期,细胞凋亡情况明显,总凋亡率显著升高(p < 0.05)。结论 0.7 mmol/L的OA刺激HepG2细胞24 h时即可建立脂毒性肝损伤模型。
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Establishment of a Model of Lipotoxic Injury Induced by Oleate in HepG2 cells
Abstract:Objective To investigate the effect of oleic acid (OA) on lipid metabolism in HepG2 cells and establish a model of lipotoxicity damage in HepG2 cells. Methods HepG2 cells were stimulated with OA at different concentrations (0、0.1、0.3、0.5、0.7、0.9 mmol/L) for 24 h. Cell proliferation was detected by cck-8 colorimetric assay. Cytotoxicity was detected by LDH release method. Staining and kitsEstablishment of a Model of Lipotoxic Injury Induced by Oleate in HepG2 cells were used to observe the lipid deposition of cells, and to measure intracellular triglyceride (TG), malondialdehyde (MDA), superoxide dismutase (SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in culture medium. Levels were used to determine the optimal model concentration; cell cycle assays were performed using the cell cycle assay kit, Hoechst 33342 staining and Annexin-V FITC-PI flow cytometry were used to detect apoptosis. Results When HepG2 cells were stimulated with 0.7 mmol/L OA for 24 h, the cell proliferation level was inhibited to some extent (p < 0.05), LDH cytotoxicity was at a low level, lipid deposition was significant (p < 0.05), ALT, AST, MDA content levels were increased significantly (p < 0.05), the activity of SOD was decreased significantly (p < 0.05), the cell cycle was inhibited in the G1/S phase, the apoptosis was obvious, and the total apoptosis rate was significantly increased (p < 0.05). Conclusion The fatty acid liver injury model can be established when 0.7 mmol/L OA stimulates HepG2 cells for 24 h.
Keywords: Lipotoxicity Oleate Non-Alcoholic Fatty Liver Disease
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