27-羟基胆固醇对神经细胞和星形胶质细胞溶酶体胆固醇转运相关因子表达的影响

The influence of 27-hydroxycholesterolon cholesterol transport associated factor in co-cultured SH-SY5Y cells and C6 cells

目的探讨27-羟基胆固醇(27-hydroxycholesterol,27-OHC)在神经细胞(SH-SY5Y)和星形胶质细胞(C6)共培养体系中对细胞溶酶体胆固醇转运相关因子表达的影响。方法采用Trans-well小室进行SH-SY5Y细胞和C6细胞共培养,使用不同剂量的27-OHC对共培养体系下细胞进行处理,分为对照组(DMEM组)、低剂量组(27-OHC 5μmol/L)、中剂量组(27-OHC 10μmol/L)及高剂量组(27-OHC 20μmol/L)。采用COD-PAP法检测两种细胞中的总胆固醇浓度;采用Real-time PCR和Western blotting检测两种细胞溶酶体内特异性胆固醇转运蛋白质——尼曼匹克蛋白质1(Niemann-Pick C protein 1,NPC1)、NPC2的基因表达水平和蛋白质表达水平。结果随着27-OHC处理浓度的升高,SH-SY5Y细胞内胆固醇浓度逐渐上升(P <0. 05),C6细胞内胆固醇浓度逐渐降低(P <0. 05); SH-SY5Y细胞溶酶体内NPC基因及蛋白质表达水平显著下调(P <0. 05); C6细胞内溶酶体NPC基因及蛋白质表达水平显著上调(P <0. 05)。结论 27-OHC可引起神经细胞及星形胶质细胞溶酶体内胆固醇转运障碍,使胆固醇在神经细胞溶酶体内蓄积。

Objective To investigate the effect of 27-hydroxycholesterol(27-OHC)on cell lysosomal cholesterol transport associated factor under the conditions of co-culture of nerve cells and astrocytes.Methods SH-SY5 Y cells and C6 cells were co-cultured with transwell chamber.The experiment was divided into control group(serum-free medium),27-OHC 5μmol/L group(low dose group),27-OHC 10μmol/L group(middle dose group)and 27-OHC 20μmol/L group(high dose group).The total cholesterol levels were detected by total cholesterol kit.The expression level of mRNA and proteins of lysosomal cholesterol transport associated proteins(including NPC1 and NPC2)were detected by real-time PCR and Western blotting.Results The total cholesterol levels of cellular protein from the C6 cells treated with 5,10 and 20μmol/L 27-OHC was significantly decreased compared with control(P<0.05).However,SH-SY5 Y cells in 27-OHC treatment groups had higher total cholesterol levels(P<0.05).Results showed reduced levels of lysosome cholesterol associated genes and proteins(NPC1 and NPC2)in SH-SY5 Y cells(P<0.05)and increased levels in C6 cells(P<0.05).Conclusion Our data indicate that 27-OHC causes neuronal and astrocyte lysosomal cholesterol transport disorders,and accumulate lysosomal cholesterol in nerve cells.