腺病毒介导的小鼠Flt3配体抗肝癌基因治疗的研究  

Gene transfer of murine Flt3 ligand mediated by adenoviral vector efficiently induces growth inhibition of murine liver cancer

作　　者：杨庆[1] 杨广顺[1] 卫立辛[1] 贾凤岐[1] 吴孟超[1] 郭亚军 

机构地区：[1]第二军医大学东方肝胆外科研究所肿瘤免疫与基因治疗中心,上海200438 [2]国际合作肿瘤研究所

出　　处：《中华医学杂志》2002年第11期775-779,共5页National Medical Journal of China

基　　金：上海市卫生系统百名优秀学科带头人培养基金资助项目 (3 119975 0 3 0 2 10 40 3 )

摘　　要：目的　观察腺病毒载体介导的小鼠Flt3配体 (mFL)对小鼠肝癌的基因治疗效果。方法　腺病毒体外感染小鼠肝癌细胞株Hepa1 6 ,4 8h后 ,用绿色荧光蛋白的表达检测感染的效率 ,ELISA检测培养上清中mFL的表达量 ,每天细胞计数 (连续 14d)观察细胞的生长曲线。建立小鼠肝癌模型 ,肿瘤局部分别单次注射 1× 10 9表达形成单位的mFL重组腺病毒、空载体对照和PBS ,观察肿瘤大小和小鼠生存期。治疗 2 0d后 ,取肿瘤消退小鼠的脾细胞过继至正常小鼠 ,3d后接种Hepa1 6细胞 ,观察肿瘤大小 ;治疗 38d后 ,再接种Hepa1 6细胞或EL4细胞对照于肿瘤消退小鼠原接种部位的对侧 ,观察肿瘤大小并作统计学处理。结果　腺病毒体外能有效地感染靶细胞Hepa1 6 ,培养上清中mFL表达量为 80 .5ng·10 -6·2 4h-1± 7.3ng·10 -6·2 4h-1,感染后Hepa1 6的生长未受到明显抑制。瘤体内注射mFL重组腺病毒导致 90 %的小鼠肿瘤消退 ,并显著延长小鼠生存期 ;脾细胞过继能保护正常小鼠免于Hepa1 6细胞的攻击 ;再接种Hepa1 6细胞于肿瘤消退小鼠 ,未见肿瘤生长 ,而再接种的EL4细胞呈渐进性生长 ,与对照组无显著差异。结论　腺病毒介导的小鼠Flt3配体的基因治疗导致肿瘤消退 ,并能激发可过继的、特异性免疫保护反应 ,可能成为有效的肝癌基因治疗的候?Objective To observe the in vivo therapeutic effects of murine Flt3 ligand (mFL) mediated by recombinant adenoviral vector on murine liver cancer. Methods Murine liver cancer cell line Hepal 6 was infected with adenovirus in vitro. The infection efficacy was measured by green fluorescence protein (GFP) expression and the amount of mFL in supernatant was measured by ELISA 48 hrs following infection of Hepa1 6. AdmFL, Ad null, and PBS were added into the culture of Hepa1 6 cells, the number of cells was counted every other day for 14 days. A murine liver cancer model was established by subcutaneous inoculation of Hepa1 6 cells. A single dosage of 1×10 9 expression forming unit (efu) of Ad mFL, Ad null or PBS was injected intratumorally. The tumor volume and survival rate were measured twice a week. Twenty days after treatment of adenoviral vectors, three treated mice with their tumor dissapearing were killed and their spleen was taken. The splenocytes from these tumor free mice were adoptively transferred to naive mice to whom Hepa1 6 cells were inoculated 3 days thereaftter and then the tumor volume was measured once a week.for 4 weeks. 38 days after administration of adenoviral vectors, tumor free animals were rechallenged by parental Hepa1 6 cells or syngenic EL4 lymphoma cells at the opposite sites of the original inoculation sites, and the tumor volume was also measured once a week for 4 weeks. Results Adenoviral vector efficiently infected Hepa1 6 cells in intro, and lead to the secretion of high levels of mFL protein (80.5±7.3 ng/10 6/24 h) in the supernatant. The growth of Hepa1 6 tumor was significantly inhibited by one single intratumoral administration of Ad mFL in 90% of the treated mice, and the tumor gradually grew in the two other groups. Two of the 7 mice (30%) in PBS group died in 17 days, 14% still lived in 37 days, and all died within 45 days. The mice in the Ad null group began to die since the 21 st day, only 11% of them were still alived in 60 days. All mice in the

关 键 词：腺病毒介导 小鼠 FLT3配体 肝细胞癌 基因疗法 造血细胞生长因子 配体 

分 类 号：R735.705[医药卫生—肿瘤]