荧光素酶标记胶质瘤细胞模型的构建及其在溶瘤病毒治疗中的应用  

Establishment of the glioma cell model marked by luciferase and study of its application in oncolytic virus therapy research

作　　者：胡跃东 张梦梦 张俊文 米蕊芳[1] Hu Yuedong;Zhang Mengmeng;Zhang Junwen;Mi Ruifang(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China)

机构地区：[1]首都医科大学,北京市神经外科研究所,北京100070

出　　处：《中华神经外科杂志》2022年第8期843-848,共6页Chinese Journal of Neurosurgery

基　　金：北京市自然科学基金(7182026,7222019)。

摘　　要：目的:构建荧光素酶标记的患者来源胶质瘤细胞模型,并探讨其在溶瘤1型单纯疱疹病毒(oHSV-1)及溶瘤腺病毒(oAD)治疗中的应用。方法:显微镜下观察前期建立的患者来源胶质瘤细胞(命名为U022细胞)的形态特征;通过胶质纤维酸性蛋白(GFAP)免疫荧光实验对患者来源U022细胞进行鉴定;通过CCK-8实验测定细胞增殖曲线(n=4);通过慢病毒感染及嘌呤霉素筛选方法获得携带荧光素酶标记的患者来源U022细胞,设置阴性对照组和荧光素酶慢病毒感染组;通过活体成像系统(IVIS)及CCK-8实验测定oHSV-1和oAD对患者来源U022细胞的作用,根据不同的病毒感染复数(0、0.001、0.01、0.1、1、10,每组n=4)的梯度进行分组。结果:患者来源U022细胞符合胶质瘤细胞的形态特征;细胞增殖曲线分析显示,U022细胞增殖较快,细胞倍增时间为19.2 h;进一步行免疫荧光实验显示U022细胞中GFAP呈阳性表达。经嘌呤霉素筛选成功获得荧光素酶标记的胶质瘤细胞(U022-luc),并且荧光素酶发光实验显示细胞的荧光强度随着细胞数量的增加而增强,呈线性正相关(R2=0.97,P<0.001)。IVIS生物素发光实验及CCK-8实验检测显示,oHSV-1和oAD均对U022-luc细胞有显著的毒性作用,并且随着滴度的增加,病毒的作用加强(均P<0.01);当病毒感染复数等于10时,oHSV-1对U022-luc细胞的抑制率分别为(68.77±5.96)%和(66.27±4.89)%,而oAD对U022-luc细胞的抑制率分别为(64.34±3.13)%和(69.73±4.87)%。结论:体外可成功构建荧光素酶标记的患者来源胶质瘤细胞模型;该细胞模型可应用于oHSV-1和oAD治疗的定量研究中。Objective To establish the patient-derived glioma cell model marked by luciferase in vitro,and to further investigate its application in oncolytic herpes simplex virus 1(oHSV-1)or oncolytic adenovirus(oAD)therapy research.Methods The patient-derived U022 glioma cells established previously were observed under the microscope for study of their morphological features.The U022 cells were identified by glial fibrillary acidic protein(GFAP)immunofluorescence staining.Cell proliferation rate was detected by CCK-8 method(n=4).Glioma cells carrying luciferase were obtained by lentivirus infection following puromycin selection.The negative control and the luciferase lentivirus infected(Luc)groups were set up during this process.The cell survival rates under oHSV-1 or oAD were detected and analyzed by In Vivo Imaging System(IVIS)and CCK8 assay,and those experiments were grouped according to the gradient of multiplicity of infection(MOI,MOI=0,0.001,0.01,0.1,1,10,n=4)of different viruses.Results Patient-derived U022 cells had the typical morphological features of glioma cells.And cell proliferation curve of U022 cells showed that the cells proliferated quickly and the doubling time of them was 19.2 h.Immunofluorescence staining showed that GFAP expression was positive in U022 cells.After that,U022-luc cells carrying luciferase gene were successfully obtained through lentivirus infection and puromycin selection,and fluorescein luminescence assay showed that cell fluorescence intensity increased with the enrichment of cell numbers,which indicated a positive linear regression(R2=0.97,P<0.001).IVIS and CCK-8 analysis showed that both the herpes simplex virus 1(oHSV-1)and the oncolytic adenovirus(oAD)had significant inhibitory effect on U022-luc glioma cells.Moreover,the efficiency was increased following the rise of the virus titer(both P<0.010).When the multiplicity of infection reached 10,the inhibition effect of oHSV-1 on U022-luc cells detected by IVIS and CCK-8 were up to(68.77±5.96)%and(66.27±4.89)%respectively.Meanwhile

关 键 词：神经胶质瘤 溶瘤病毒 荧光素酶 溶瘤1型单纯疱疹病毒 溶瘤腺病毒 

分 类 号：R739.41[医药卫生—肿瘤]