| 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、50.0滋M）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，Westernblot法检测磷酸化AKT（p-AKT）、cleaved-caspase-9蛋白表达，AnnexinV-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、50.0滋M27-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（3.125~50.0滋M）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（Ser473）、cleaved-caspase-9（37/35kD）表达明显增强；经LY294002预作用后，不同浓度（0~50.0滋M）27-OHCH处理组p-AKT（Ser473）表达均受抑制，而12.5、25.0、50.0滋M27-OHCH处理组cleaved-caspase-9蛋白表达变化不明显。给药48h，12.5、25.0滋M27-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。 |
| 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 |
| DOI：10.12056/j.issn.1006-2785.2018.40.18.2018-687 |
| 分类号: |
| 基金项目:浙江省科技计划项目（2014C33132） |
|
| 27-hydroxycholesterol induces apoptosis of human neuroblastoma cells through AKT signaling pathway |
|
|
|
Affiliated Zhoushan Hospital of Wenzhou Medical University
|
| Abstract: |
| Objective To investigate the mechanism of apoptosis induced by 27-hydroxycholesterol (27-OHCH) in human neuroblastoma cells (SH-SY5Y ). Methods SH-SY5Y cells were treated with 27-OHCH of different concentrations (0, 3.125, 6.25, 12.5, 25, 50滋M). 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-SY5Y cells in 27-OHCH treatment groups(12.5, 25 and 50滋M for 24h) were significantly lower than that of control group (P＜0.05), while when the treatment lasted for 48h the cell survival rate were lower than that of control group in all 27-OHCH concentration groups (3.125-50滋M) (P＜0.05). The light microscope showed that 27-OHCH induces the apoptosis of SH-SY5Y cells and reduces the cells number, while inhibitor LY294002 weakened the cytotoxicity of 27-OHCH. When SH-SY5Y cells treated with 27-OHCH (3.125-50滋M) for 48h, the expression levels of p-AKT (Ser 473) and cleaved caspase-9 were increased. When SH-SY5Y 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 in 12.5, 25.0 and 50滋M 27-OHCH treatment groups had no significant difference. Flow cytometry showed that LY294002 attenuated apoptosis of SH-SY5Y cells induced by 27-OHCH (P＜0.05). Conclusion 27-OHCH induces apoptosis of SH-SY5Y cells through AKT signaling pathway. |
| Key words: 27-hydroxycholesterol SH-SY5Y cells Apoptosis AKT signaling pathway |
