27-羟基胆甾醇诱导人神经母细胞瘤细胞凋亡的调控机制  

27-hydroxycholesterol induces apoptosis of human neuroblastoma cells through AKT signaling pathway

作　　者：朱梦娇 黄燕燕 曹立[3] 唐维国 ZHU Mengjiao;HUANG Yanyan;CAO Li(Department of Neurology,Zhoushan Hospital Affiliated to Wenzhou Medical University,Zhoushan316021,China)

机构地区：[1]温州医科大学附属舟山医院神经内科,316021 [2]温州医科大学附属舟山医院细胞分子生物学实验室,316021 [3]上海交通大学医学院附属瑞金医院神经内科

出　　处：《浙江医学》2018年第18期1996-2000,2012,共6页Zhejiang Medical Journal

基　　金：浙江省科技计划项目(2014C33132)

摘　　要：目的探讨27-羟基胆甾醇(27-OHCH)诱导人神经母细胞瘤细胞(SH-SY5Y)凋亡的调控机制。方法不同浓度(0、3.125、6.25、12.5、25.0、50.0μM)的27-OHCH作用SH-SY5Y细胞,采用CCK-8法检测细胞存活率,在光学显微镜下观察27-OHCH处理组、添加苏氨酸蛋白激酶(AKT)抑制剂(LY294002)的27-OHCH处理组细胞形态变化,Western blot法检测磷酸化AKT(p-AKT)、cleaved-caspase-9蛋白表达,Annexin V-PE/7-AAD双染流式细胞仪检测细胞凋亡情况。结果给药24h,12.5、25.0、50.0μM 27-OHCH处理组SH-SY5Y细胞存活率均明显降低(均P<0.05);给药48h,不同浓度(3.125~50.0μM)27-OHCH处理组SH-SY5Y细胞存活率均明显降低(均P<0.05)。在显微镜下观察发现,27-OHCH能诱导SH-SY5Y细胞凋亡,使细胞数量减少,而LY294002能减弱27-OHCH诱导的细胞毒效应。随着27-OHCH处理组浓度的增加,SH-SY5Y细胞内p-AKT(Ser 473)、cleaved-caspase-9(37/35k D)表达明显增强;经LY294002预作用后,不同浓度(0~50.0μM)27-OHCH处理组p-AKT(Ser 473)表达均受抑制,而12.5、25.0、50.0μM 27-OHCH处理组cleaved-caspase-9蛋白表达变化不明显。给药48h,12.5、25.0μM 27-OHCH处理组均能引起SH-SY5Y细胞凋亡效应,而LY294002预作用2h能逆转细胞凋亡效应(均P<0.05)。结论 27-OHCH可通过AKT信号通路诱导SH-SY5Y细胞凋亡。Objective To investigate the mechanism of apoptosis induced by 27-hydroxycholesterol（27-OHCH） in human neuroblastoma cells（SH-SY5 Y）. Methods SH-SY5 Y cells were treated with 27-OHCH of different concentrations（0, 3.125, 6.25,12.5, 25, 50μM）. The cell viability was detected by CCK-8 assay and cell apoptosis was detected by Annexin V-PE/7-AAD. The effects of 27-OHCH, AKT inhibitor LY294002 and 27-OHCH co-treatment on cell morphology were observed under the light microscope. Western blot was used to detect the expression level of phosphorylated AKT and apoptotic-associated proteins caspase-9 and cleaved-caspase-9. Results The survival rate of SH-SY5 Y cells in 27-OHCH treatment groups（12.5, 25 and50μM for 24 h） were significantly lower than that of control group（P〈0.05）, while when the treatment lasted for 48 h the cell survival rate were lower than that of control group in all 27-OHCH concentration groups（3.125-50μM）（P〈0.05）. The light microscope showed that 27-OHCH induces the apoptosis of SH-SY5 Y cells and reduces the cells number, while inhibitor LY294002 weakened the cytotoxicity of 27-OHCH. When SH-SY5 Y cells treated with 27-OHCH（3.125-50μM） for 48 h, the expression levels of p-AKT（Ser 473） and cleaved caspase-9 were increased. When SH-SY5 Y cells were pre-treated with LY294002, the expression level of p-AKT（Ser 473） was inhibited in all 27-OHCH treatment groups, while the protein expression level of cleaved-caspase-9 in12.5, 25.0 and 50μM 27-OHCH treatment groups had no significant difference. Flow cytometry showed that LY294002 attenuated apoptosis of SH-SY5 Y cells induced by 27-OHCH（P〈0.05）. Conclusion 27-OHCH induces apoptosis of SH-SY5 Y cells through AKT signaling pathway.

关 键 词：27-羟基胆甾醇 人神经母细胞瘤细胞 细胞凋亡 AKT信号通路 

分 类 号：R739.4[医药卫生—肿瘤]