脑脂结合蛋白在胶质母细胞瘤U251细胞自噬中的调节作用  

Brain lipid binding protein regulates autophagy in U251 glioblastoma cell line

作　　者：张惠雯 张俊荣[2] 施金洪 ZHANG Huiwen;ZHANG Junrong;SHI Jinhong(Department of Nursing,the Affiliated Hospital of Nantong University,Nantong 226001;Department of Gynaecology and Obstetrics,the Affiliated Hospital of Nantong University,Nantong 226001;Department of Human Anatomy,Medical School,Nantong University)

机构地区：[1]南通大学附属医院护理部,南通226001 [2]南通大学附属医院妇产科,南通226001 [3]南通大学医学院人体解剖学系

出　　处：《南通大学学报（医学版）》2023年第3期207-210,共4页Journal of Nantong University(Medical sciences)

基　　金：南通大学医学院自然科学基金项目(TDYX2022014)。

摘　　要：目的:探讨脑脂结合蛋白(brain lipid binding protein,BLBP)在胶质母细胞瘤U251细胞自噬中的调节作用。方法:应用CRISPR/Cas9技术敲除U251细胞中BLBP的表达,检测BLBP敲除后U251细胞的活力变化;Ad-GFP-LC3b腺病毒感染U251细胞后,检测细胞中GFP-LC3b绿色荧光颗粒的数目;Western Blot检测自噬相关分子微管相关蛋白1轻链3βⅠ/Ⅱ(microtubule associated protein 1 light chain 3 betaⅠ/Ⅱ,LC3bⅠ/Ⅱ)、自噬相关基因5(autophagy-related genes 5,ATG5)、选择性自噬接头蛋白(sequestosome1,SQSTM1)的蛋白表达水平;Real-time PCR检测ATG5、SQSTM1的基因表达变化。结果:BLBP基因敲除后,U251细胞的活力明显下调,GFP-LC3b绿色荧光颗粒数目较对照组明显增多;此外,LC3bⅡ的蛋白表达水平较对照组明显增高,SQSTM1蛋白水平明显下降,而ATG5的基因和蛋白表达水平均明显上调。结论:BLBP基因敲除能提高U251细胞的自噬水平,BLBP具有调节胶质母细胞瘤细胞自噬的能力。Objective:To clarify the function of brain lipid binding protein(BLBP)in cellular autophagy in U251 glioblastoma cell line.Methods:BLBP expression was deleted by using CRISPR/Cas9 technology,and then the cells viability was investigated.After treatment of Ad-GFP-LC3b,the GFP-LC3b puncta number was measured.The proteins level of autophagy-related markers microtubule associated protein 1 light chain 3 betaⅠ/Ⅱ(LC3bⅠ/Ⅱ),autophagy-related genes 5(ATG5),sequestosome1(SQSTM1)were determined by Western Blot,while the gene levels of ATG5,SQSTM1 were detected by Real-time PCR.Results:In BLBP knockout cells,the cells viability was decreased,but the number of GFP-LC3b puncta was increased.The protein level of LC3bⅡwas elevated in BLBP knockout U251 cells than that in control cells,but SQSTM1 was downregulated;both protein and gene level of ATG5 were upregulated in BLBP knockout cells.Conclusion:BLBP deficiency in U251 cells promotes the cellular autophagy,and BLBP can regulate the autophagy in glioblastoma cells.

关 键 词：胶质母细胞瘤 脑脂结合蛋白 U251细胞 自噬 

分 类 号：R739.41[医药卫生—肿瘤]