Rab32对小鼠肝细胞系AML12细胞内脂质代谢的影响

目的 探讨调控Rab32蛋白表达对小鼠AML12细胞内脂质代谢的影响.方法 常规培养AML12细胞,通过转染FCD-EYFP-Rab32慢病毒表达载体构建Rab32过表达细胞系(过表达组) ,利用CRISPR/Cas9基因敲除技术构建Rab32敲除细胞系(敲除组) ,正常细胞设为对照组.Western blot检测各组细胞中Rab32蛋白表达水平;尼罗红染色后,激光共聚焦显微镜观测各组细胞内的脂滴数量和面积;定量试剂盒检测各组细胞内甘油三酯及总胆固醇水平,流式细胞术检测细胞凋亡水平.结果 与对照组比较,过表达组AML12细胞中Rab32蛋白表达水平显著上调,而Rab32敲除组细胞中Rab32蛋白表达水平显著下调(P＜ 0.01);过表达组AML12细胞内脂滴数量过表达组vs对照组: (21.91 ±14.32) vs (33.89 ± 17.31) ,P＜ 0.01]及面积过表达组vs对照组: (16.82 ± 14.37) μm2 vs (22.27 ±14.10) μm2,P＜ 0.01]显著减少,每毫克蛋白甘油三酯过表达组vs对照组: (104.03 ± 12.28) nmol vs(130.94 ± 4.21) nmol,P＜ 0.01]及胆固醇水平过表达组vs对照组: (46.92 ± 1.26) μg vs (81.11 ±0.65) μg,P＜ 0.01]显著降低;而Rab32敲除组细胞内脂滴数量(58.23 ± 42.28) ]及面积(53.31 ±36.33) μm2]较对照组显著增加(P＜ 0.01) ,其每毫克蛋白甘油三酯(159.03 ± 8.85) nmol]及胆固醇水平(93.38 ± 3.33) μg]显著增高(P＜ 0.01) .3组细胞凋亡水平差异无统计学意义(P＞ 0.05) .结论 Rab32促进小鼠肝细胞AML12胞内脂质代谢.

Effect of Rab32 on lipid metabolism in mouse hepatic AML12 cells

Objective To determine the effect of regulation of Rab32 on the lipid metabolism in murine hepatic alpha mouse liver 12 (AML12) cells. Methods Lentivirus expressing FCD-EYFP-Rab32was transfected into AML12 cells for overexpression of Rab32, and CRISPR/Cas9 gene knockout technology was used to knockout Rab32 in the cells. Untreated AML12 cells were used as normal control. The protein level of Rab32 was determined by Western blotting. The intracellular lipid droplets were observed by confocal microscopy after Nile red fluorescent staining. The contents of intracellular triglyceride (TG) and total cholesterol (TC) were measured by corresponding quantitation kits. Cell apoptosis was assayed by flow cytometry. Results Compared with the normal control cells, the protein level of Rab32 was significantly increased in the overexpressed cells but decreased in the knockout cells (P＜0.01). The number (21.91 ±14.32 vs 33.89 ± 17.31, P＜0.01) and size (16.82 ± 14.37 vs 22.27 ± 14.10 μm2, P＜0.01) of lipid droplets per cell were significantly decreased in the overexpressed cells when compared with the untreated cells, while the intracellular contents of TG (104.03 ± 12.28 vs 130.94 ± 4.21 nmol, P＜0.01) and TC (46.92 ± 1.26 vs 81.11 ± 0.65 μg, P＜0.01) were decreased. Whereas, the knockout cells had larger lipid droplet number (58.23 ± 42.28, P＜0.01) and total area (53.31 ± 36.33 μm2, P＜0.01), and higher intracellular TG (159.03 ± 8.85 nmol, P＜0.01) and TC (93.38 ± 3.33 μg, P＜0.01). No obvious difference was found in cell apoptosis among the 3 groups of cells (P＞ 0.05). Conclusion Rab32 promotes the lipid metabolism in the AML12 cells, and the molecule may be regarded as a new target in treatment of nonalcoholic fatty liver disease.