Netrin-1通过下调氧糖剥夺诱导的人神经母细胞瘤自噬促进细胞存活  

Netrin-1 protects SH-SY5Y neuroblastoma cells from oxygen-glucose deprivation by attenuating autophagy

作　　者：阙嘉丽 李晨光[1] 刘可嘉[1] 余剑[1] 

机构地区：[1]中山大学附属第一医院神经科,卫生部国家临床重点专科,教育部国家重点专科,广东广州510080

出　　处：《中风与神经疾病杂志》2016年第6期513-516,共4页Journal of Apoplexy and Nervous Diseases

基　　金：国家自然科学基金(No.81371276);广东省自然科学基金(No.2014A030313065);广东省重大神经疾病诊治研究重点实验室(No.2014B030301035)

摘　　要：目的通过建立体外人神经母细胞瘤(SH-SY5Y)氧糖剥夺(oxygen and glucose deprication,OGD)模型模拟脑梗死后神经元缺血缺氧环境(OGD),探索神经导向因子Netrin-1是否影响OGD后细胞的生存,以及自噬在其中的可能作用。方法体外培养SH-SY5Y细胞,检测Netrin-1对细胞活性及自噬的影响。使用自噬诱导剂雷帕霉素、自噬抑制剂3-甲基腺嘌呤干预自噬水平,同时构建过表达Netrin-1受体UNC5H2-HA基因的HEK293T细胞;CCK-8试剂盒检测细胞活性,免疫印迹检测自噬相关标志物(Beclin 1、LC3、p62)的表达及免疫荧光检测LC3斑点的形成。结果与OGD对照组相比,Netrin-1及3-甲基腺嘌呤的干预均显著提高细胞活性,伴有LC3-Ⅱ斑点形成减少;相反,雷帕霉素增加LC3-Ⅱ斑点的同时抑制细胞活性,并减弱了Netrin-1提高细胞活性的作用。过表达UNC5H2-HA细胞较对照质粒组相比,LC3-Ⅱ表达增加;同时进行Netrin-1干预后仅过表达组出现LC3-Ⅱ表达下降,p62表达上升。结论 Netrin-1可能通过受体UNC5H2下调OGD损伤的人神经母细胞瘤自噬而提高细胞活性。Objective To mimic hypoxic and ischemia environment caused by stroke,in vitro oxygen-glucose deprivation（ OGD） model was established. The aim is to explore whether axon guidance factor Netrin-1 protectes SH-SY5 Y neuroblastoma cells from OGD by regulating autophagy. Methods SH-SY5 Ys were pretreated with Netrin-1 before OGD,apply applied autophagy inducer rapamycin and autophagy inhibitor 3-methyladeni-ne（ 3-MA） to modulate the autophagy flow,and transient transfected HEK 293 T cell with UNC5H2-HA plasmid; cell viability was examined in CCK8 assay,western Western blot detected the autophagy markers（ Beclin1,LC3,p62） and immunofluorescence detected punctuate LC3.Results We found that Netrin-1 improved cell viability and decreased the expression of LC3-Ⅱ,a protein related to autophagosome formation,in a manner sensitive to RAPA co-treatment. Additionally,the ability of Netrin-1 to decrease the expression of LC3-Ⅱ was present in UNC5H2-overexpressing HEK 293 T cells but not vehivle-transfected cells,indicated the importance of Netrin-1-UNC5H2 interaction to this effect. Conclusion These results indicate that Netrin-1 may improve cell survival by attenuat-ing autophagy via UNC5H2.

关 键 词：NETRIN-1 氧糖剥夺 自噬 UNC5H2 

分 类 号：R739.4[医药卫生—肿瘤]