EA-IL-15溶瘤腺病毒的构建及其对胶质瘤细胞特异性及有效性研究  

Construction of the EA-IL-15 Oncolytic Adenovirus and Investigation on its Specificity and Efficacy in Glioma Cells

作　　者：季佳宇 王启俨 方胜 田毅夫 刘福生[1] 米蕊芳[1] Ji Jiayu;Wang Qiyan;Fang Sheng(Brain Tumor Research Center,Beijing Neurosurgical Institute,Beijing Tiantan Hospital Affiliated to Capital Medical University,Beijing 100070,China)

机构地区：[1]北京市神经外科研究所脑肿瘤研究中心、首都医科大学附属北京天坛医院,100070

出　　处：《医学研究杂志》2020年第10期47-51,共5页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81672478);北京市自然科学基金资助项目(7182026)。

摘　　要：目的构建插入EA-IL-15双基因的溶瘤腺病毒(oAD-EA-IL-15),探讨其在体外对胶质瘤细胞的杀伤效果和特异性。方法PCR法将EA-IL-15双基因插入腺病毒穿梭载体pXC1,将穿梭质粒pXC1-EA-IL-15及辅助质粒pBHG loxΔE1,3 Cre质粒共转染HEK393细胞,获得溶瘤腺病毒oAD-EA-IL-15。通过CCK-8细胞毒性实验,测定细胞相对活性,绘制剂量反应曲线。结果成功构建溶瘤腺病毒载体(oAD)及含EA-IL-15双基因的溶瘤腺病毒载体(oAD-EA-IL-15);体外包装获得相应的溶瘤腺病毒oAD和oAD-EA-IL-15;oAD对小鼠胶质瘤细胞GL261有明显杀伤作用(P<0.05),对小胶质细胞BV2(非肿瘤细胞)的杀伤作用明显低于对GL261(P<0.05),说明oAD具有特异性;oAD-EA-IL-15和oAD分别感染GL261、BV2细胞,对于GL261,oAD-EA-IL-15的半抑制浓度(IC 50)值为0.58×107±1.30×105 PFU/ml较oAD的IC 50值1.13×107±1.98×105PFU/ml低,对肿瘤细胞的杀伤作用强于oAD(P<0.01),对于BV2,oAD-EA-IL-15对其活性的影响低于oAD(P<0.01),说明oAD-EA-IL-15病毒的特异性优于oAD病毒。结论携带EA-IL-15双基因的溶瘤腺病毒在体外对胶质瘤细胞具有明显的杀伤作用和特异性,可为胶质瘤治疗提供更加特异有效的途径。Objective To construct an oncolytic adenovirus carrying Endo-Angio(EA)and IL-15 double genes,and further investigate its specificity and cytotoxic effect to glioma cells in vitro.Methods EA and IL-15 genes were firstly obtained by PCR method,and inserted into the shuttle vector pXC1 through gene cloning approach.Then the shuttle plasmid pXC1-EA-IL-15 and the auxiliary plasmid pBHG loxΔE1,3 Cre plasmid were cotransfected into the HEK293 cells to form the oAD-EA-IL-15 adenovirus.The specificity and cytotoxic effect of the adenoviruses were evaluated by CCK-8 method and analyzed to draw the dose response curve according to the results.Results The oncolytic adenovirus vector(oAD)and the EA-IL-15 double gene(oAD-EA-IL-15)adenovirus vector were successfully constructed,and the corresponding adenoviruses were successfully obtained by virus packaging in vitro.oAD showed significantly inhibition effect on mouse glioma cells GL261 compared to the control group(P<0.05),while the cytotoxicity effect to microglia BV2(non-tumor cells)was significantly weaker compared to GL261 group(P<0.05)which indicated the specificity of oAD.Furthermore,we detected the efficacy of oAD-EA-IL-15 and oAD on GL261 and BV2 cells respectively.The results showed oAD-EA-IL-15 had a stronger cytotoxicity effect then oAD on GL261 cells(P<0.01)and the half-inhibitory concentration(IC 50)value of oAD-EA-IL-15(0.58×107±1.30×105 PFU/ml)was lower than the IC 50 value of oAD(1.13×107±1.98×105 PFU/ml).Moreover,the cytotoxicity effect of the oAD-EA-IL-15 to BV2 cells was weaker than oAD(P<0.01),indicating oAD-EA-IL-15 had better specificity to glioma cells.Conclusion The oncolytic adenovirus carrying the EA-IL-15 double gene had significant specificity and cytotoxic effect in treating glioma cells in vitro,which could be a safer and more efficient approach in glioma therapy.

关 键 词：胶质瘤 溶瘤腺病毒 人内皮抑素 血管生成抑素 IL-15 

分 类 号：R73[医药卫生—肿瘤]