放射联合替莫唑胺治疗对U87-EGFRvⅢ细胞及其移植瘤影响的实验研究  

Effect of combined therapy of radiosurgery and temozolomide on U87-epidermal growth factor receptor and variant III cells and their xenograft

作　　者：林益光[1] 刘东[1] 刘晓民[2] 徐德生[1] Lin Yiguang;Liu Dong;Liu Xiaomin;Xu Desheng(Gamma Knife Centre,Department of Neurosurgery,Second Hospital of Tianjin Medical University,Tianjin 300211,China;Gamma Knife Centre,Department of Neurosurgery,Tianjin Huanhu Hospital,Tianjin 300350,China)

机构地区：[1]天津医科大学第二医院神经外科伽玛刀中心,天津300211 [2]天津市环湖医院神经外科伽玛刀中心,300350

出　　处：《中华神经医学杂志》2019年第12期1215-1223,共9页Chinese Journal of Neuromedicine

摘　　要：目的通过体外、体内实验观察放射联合替莫唑胺治疗对胶质瘤U87-表皮生长因子受体的Ⅲ型突变体(EGFRvⅢ)细胞及其移植瘤的影响。方法(1)常规培养人脑胶质瘤U87-EGFRvⅢ细胞,CCK-8实验检测不同浓度的替莫唑胺对U87-EGFRvⅢ细胞增殖的影响,计算替莫唑胺的半抑制浓度(IC50);将细胞分为对照组、6 Gy放射治疗组、12 Gy放射治疗组,分别以0、6、12 Gy边缘剂量照射后应用Western blotting实验检测3组细胞侵袭相关蛋白和基因修复相关蛋白的表达;将细胞分为对照组、6 Gy放射治疗组、6 Gy放射联合替莫唑胺治疗组(经6 Gy剂量照射后加入5 mmol/L替莫唑胺),24 h后应用Western blotting实验检测3组细胞侵袭相关蛋白和基因修复相关蛋白的表达;(2)应用对照组、6 Gy放射治疗组、6 Gy放射联合替莫唑胺治疗组细胞悬液制作裸鼠颅内移植胶质瘤模型,移植后第7、14、21天应用生物发光成像(BLI)检测裸鼠体内荧光素酶的表达并测量移植瘤体积,绘制裸鼠的生存曲线、体质量曲线。免疫组化染色检测颅内移植胶质瘤EGFRvⅢ、EGFR、基质金属蛋白酶(MMP)-9、血管内皮生长因子(VEGF)、Ki67的表达。结果(1)替莫唑胺对U87-EGFRvⅢ细胞的IC50为5 mmol/L;与对照组比较,6 Gy放射治疗组细胞EGFRvⅢ、EGFR、MMP-2、MMP-9、VEGF、MGMT、蛋白激酶B(AKT)-1、β-链蛋白(β-catenin)和Ku70蛋白的表达增加,与6 Gy放射治疗组比较,12 Gy放射治疗组细胞EGFRvⅢ、EGFR、MMP-2、MMP-9、VEGF、6-氧-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、AKT-1、β-catenin和Ku70蛋白的表达降低,差异均有统计学意义(P<0.05);与6 Gy放射治疗组比较,6 Gy放射联合替莫唑胺治疗组细胞EGFRvⅢ、EGFR、MMP-2、MMP-9、VEGF、MGMT、AKT-1、β-catenin和Ku70蛋白的表达降低,差异均有统计学意义(P<0.05)。(2)与对照组、6 Gy放射治疗组比较,6 Gy放射联合替莫唑胺治疗组细胞的成瘤性低,移植后21 d生存率、体质量均Objective To explore the effect of radiosurgery in combination of temozolomide therapy in vitro and in vivo on invasion and endocrine function of human glioma cell line U87-epidermal growth factor receptor and variant III(EGFRvIII)and their xenograft.Methods(1)Human glioma U87-EGFRvIII cells were routinely cultured;CCK-8 was used to detect the effect of temozolomide at different concentrations on proliferation of U87-EGFRvIII cells,and the semi-inhibitory concentration(IC50)of temozolomide was calculated;the U87-EGFRvIII cells were divided into control group,6 Gy radiotherapy group and 12 Gy radiotherapy group,and the expressions of invasion-related proteins and gene repair proteins in the three groups were detected by Western blotting after 0,6 and 12 Gy edge-dose irradiation,respectively;the U87-EGFRvIII cells were divided into control group,6 Gy radiotherapy group and 6 Gy radiation combined with temozolomide treatment group(5 mmol/L temozolomide was added after 6 Gy irradiation),and 24 h after each treatment,Western blotting was used to detect the expressions of invasion related proteins and gene repair related proteins in the three groups.(2)Nude mouse transplantation intracranial glioma models were established with cell suspension of the control group,6 Gy radiotherapy group and 6 Gy radiation combined with temozolomide treatment group;7,14,21 d after transplantation,bioluminescence imaging(BLI)was employed to detect luciferase expression in nude mice in vivo and measurement of transplanted tumors was performed;survival curve and body mass curve were drew in nude mice;immunohistochemical staining was used to detect the expressions of EGFRvIII,epidermal growth factor receptor(EGFR),matrix metalloproteinase(MMP)-9,vascular endothelial growth factor(VEGF)and Ki67 in intracranial transplanted gliomas.Results(1)The IC50 of temozolomide on U87-EGFRvIII cells was 5 mmol/L;as compared with those in the control group,the expressions of EGFRvIII,EGFR,MMP-2,MMP-9,VEGF,MGMT,AKT-1,β-catenin and Ku70 significantly incre

关 键 词：表皮生长因子受体的Ⅲ型突变体 突变 放射治疗 替莫唑胺 侵袭 增殖 

分 类 号：R73[医药卫生—肿瘤]