沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨  

Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF

作　　者：黄海宁 汪汇 龙超良 张浩 汪海[2,3,4] HUANG Hai-ning;WANG Hui;LONG Chao-liang;ZHANG Hao;WANG Hai(Affiliated Hospital of Jining Medical University,Jining 272029;Thadweik Academy of Medicine,Beijing 100039;College of Heacth Science,Jiangsu Normal University,Xuzhou 221116;General Hospital of PLA,Beijing 100853,China)

机构地区：[1]济宁医学院附属医院,山东济宁272029 [2]北京赛德维康医药研究院,北京100039 [3]江苏师范大学健康科学学院,徐州221116 [4]中国人民解放军总医院,北京100853

出　　处：《中国应用生理学杂志》2022年第2期169-174,共6页Chinese Journal of Applied Physiology

基　　金：国家973计划项目子课题(2011CB518206);济宁医学院教师科研扶持基金(JYFC2019FKJ161)。

摘　　要：目的:探讨Cereblon(CRBN)对沙利度胺抑制人肺癌A549细胞及人肝癌HepG2细胞分泌VEGF/bFGF的影响。方法:采用慢病毒介导的短发夹RNA(shRNA)干扰技术建立稳定敲低CRBN的A549细胞系(A549_(CRBN))及HepG2细胞系(HepG2_(CRBN))并通过实时定量PCR(Real-time PCR)和蛋白质印记(Western blot)实验验证。将A549细胞分为阴性对照组(A549_(luciferase))、CRBN低表达组(A549_(CRBN));HepG2细胞分为阴性对照组(HepG2_(luciferase))、CRBN低表达组(HepG2_(CRBN)),以上细胞按照3×10^(5)cells/well接种到6孔板中,放入37℃,5%CO_(2)的培养箱中培养24 h,分别加入1 ml含100μmol/L沙利度胺(thalidomide组)和1 ml 1‰DMSO(control组)的培养液,继续培养24 h再行后续实验,每组设计3个复孔。MTS法检测沙利度胺对细胞增殖的影响;Real-time PCR检测VEGF、bFGF、c-jun mRNA表达,ELISA法检测VEGF、bFGF蛋白表达。结果:与对照组比较,沙利度胺在浓度为1、10、50、100μmol/L时对A549及HepG2细胞的增殖能力无显著影响(P>0.05)。与A549_(CRBN)或HepG2_(CRBN)组比较,A549_(luciferase)及HepG2_(luciferase)组分泌的VEGF及bFGF均显著降低(P<0.05)。与A549_(luciferase)或HepG2_(luciferase)细胞的对照组比较,沙利度胺可抑制A549_(luciferase)和HepG2_(luciferase)细胞的VEGF和bFGF的表达(P<0.05),而对A549_(CRBN)和HepG2_(CRBN)细胞中VEGF和bFGF的表达无显著抑制作用;与HepG2_(luciferase)细胞的对照组比较,沙利度胺可抑制HepG2_(luciferase)细胞的c-Jun表达(P<0.01),而对HepG2_(CRBN)细胞的c-Jun表达无显著抑制作用。结论:沙利度胺对A549和HepG2细胞VEGF和bFGF表达的抑制作用可能是通过CRBN介导的,而c-Jun可能是抑制作用的关键转录因子之一。Objective:To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon(CRBN).Methods:shRNA technology was used to construct the A549 cell line(A549_(CRBN))and HepG2 cell line(HepG2_(CRBN))with stable knockdown of_(CRBN),which was verified by real-time PCR and Western blot.A549 cells were divided into negative control group(A549_(luciferase))and_(CRBN) down-regulation group(A549_(CRBN));HepG2 cells were divided into negative control group(HepG2 _(luciferase))and_(CRBN) down-regulation group(HepG2_(CRBN)).The above cells were seeded into 6-well plates at 3×10^(5) cells/well,and cultured in a 37℃,5%CO_(2) incubator for 24 h.Then,1 ml medium containing 100μmol/L thalidomide(thalidomide group)and 1 ml medium containing 1‰DMSO(control group)were added respectively,and the culture was continued for 24 hours before subsequent experiments.Each group was designed with three replicate wells.The effect of thalidomide on the activity of A549 cell line was detected by MTS assay.Real-time PCR was performed to detect mRNA expression levels of VEGF,bFGF and c-jun.ELISA assay was performed to detect protein expressions of VEGF and bFGF.Results:Compared with the control group,thalidomide at the concentrations of 1,10,50 and 100μmol/L had no significant effects on the proliferation of A549 and HepG2 cells(P>0.05).VEGF and bFGF levels in the A549_(CRBN) or HepG2 _(luciferase) groups were significantly lower than those in the A549_(CRBN) or HepG2_(CRBN) groups(P<0.05).Compared with the control group of the A549 _(luciferase) or HepG2 _(luciferase),thalidomide inhibited the expressions of VEGF and bFGF in A549 _(luciferase) and HepG2 _(luciferase) cells(P<0.05),but did not inhibit the expressions of VEGF and bFGF in A549_(CRBN) and HepG2_(CRBN) cells.Compared with the control group of the HepG2 _(luciferase),thalidomide inhibited c-Jun expression in HepG2 _(luciferase) cells(P<0.01),but did not

关 键 词：沙利度胺 血管新生 细胞培养 血管内皮生长因子(VEGF) 碱性成纤维细胞生长因子(bFGF) cereblon(CRBN) 

分 类 号：R966[医药卫生—药理学]