腺病毒介导的小鼠Flt3配体抗肝癌基因治疗的研究

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

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

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 Hepal-6. AdmFL, Ad-null, and PBS were added into the culture of Hepal-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 Hepal-6 cells. A single dosage of 1 x 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 disappearing were killed and their spleen was taken. The splenocytes from these tumor free mice were adoptively transferred to naive mice to whom Hepal-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 Hepal-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 Hepal-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 Hepal-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 alive in 60 days. All mice in the Ad-mFL group were alive at the 60(th) day after tumor implantation. Adoptive transfer of splenocytes to the animals receiving Ad-mFL treatment protected effectively them against a subsequent challenge with the identical tumor cells. Rechallenge of the Ad-mFL cured mice with the parental Hepal-6 cells resulted in complete inhibition of tumor growth. The growth of inoculated EL4 lymphoma cells gradually grew equally in the controls and the experimental mice. CONCLUSION: FL gene transfer mediated by recombinant adenoviral vector has potent therapeutic effects on Hepal-6 liver cancer, and develops long-lasting specific antitumor immunity, which may become a potent cancer gene therapy candidate for further clinic application.