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重组溶瘤腺病毒Ad-CD80-TPE-GM对Hep2细胞移植瘤的杀伤效应
引用本文:胡泽斌,鲁茁壮,吴祖泽,王立生.重组溶瘤腺病毒Ad-CD80-TPE-GM对Hep2细胞移植瘤的杀伤效应[J].中国医药生物技术,2008,3(2):116-120.
作者姓名:胡泽斌  鲁茁壮  吴祖泽  王立生
作者单位:军事医学科学院放射与辐射医学研究所实验血液学研究室,北京,100850
摘    要:目的研究自行构建的重组溶瘤腺病毒Ad-CD80-TPE-GM对裸鼠Hep2细胞移植瘤的杀伤效应。 方法裸鼠皮下接种Hep2细胞悬液建立荷人喉癌动物模型。将裸鼠分为单次治疗组和连续治疗(每天1次、连续5d)组,每组据治疗药物不同再分为PBS组、携带增强型绿色荧光蛋白报告基因的复制缺陷型5型重组腺病毒(Ad-GFP)组、Ad-CD80-TPE-GM组,以PBS、Ad-GFP组为对照组。单次治疗组于治疗后4d取外周血并剥离肿瘤,分别用有限稀释法、BCA蛋白质定量试剂盒和流式细胞术检测肿瘤组织中的病毒感染滴度、总蛋白浓度和CD80表达阳性率,ELISA法检测血清和肿瘤组织中粒细胞巨噬细胞集落刺激因子(GM-CS)表达水平,并计算肿瘤组织中病毒感染滴度量和总蛋白中GM-CSF的表达量。连续治疗组于首次给药后观察裸鼠肿瘤体积的变化,待对照组肿瘤直径〉1cm,或肿瘤表面出现明显溃疡时剥离肿瘤,计算Ad-CD80-TPE-GM组分别与2个对照组相比的相对肿瘤增殖率和抑瘤率。 结果单次治疗组中,Ad-CD80-TPE-GM组肿瘤组织中病毒感染滴度量(IU/tumor)为1.13×10^6(1.40×10^5~7.25×10^7),明显高于Ad-GFP组1.17×10^2(7.80×10^1~2.40×10^2),P=0.01],而PBS组未检测到病毒;GM-CSF表达量(pg/mg)为397.32±179.67,明显高于PBS组(4.02±0.93,P〈0.01)和Ad-GFP组(6.92±2.79,P〈0.01);CD80表达阳性率为31.6%9.0%,而PBS和Ad-GFP组均基本不表达(均P〈0.001)。连续治疗组中,Ad-CD80-TPE-GM组与PBS和Ad-GFP组相比,相对肿瘤增殖率分别为31.8%(P〈0.05)、25.9%(P〈0.01);抑瘤率分别为73.4%、77.9%(均P〈0.001)。 结论Ad-CD80-TPE-GM能在端粒酶阳性的人喉癌Hep2细胞移植瘤组织内大量复制并有效表达目的基因,具有明显的抗肿瘤效应。

关 键 词:腺病毒科  DNA  重组  粒细胞巨噬细胞集落刺激因子  抗原  CD80  基因  肿瘤抑制
收稿时间:2007-12-19
修稿时间:2007年12月19

Antitumor effect of recombinant oncolytic adenovirus Ad-CD80-TPE-GM in Hep2 xenograft tumor model
HU Ze-bin,LU Zhuo-zhuang,WU Zu-ze,WANG Li-sheng.Antitumor effect of recombinant oncolytic adenovirus Ad-CD80-TPE-GM in Hep2 xenograft tumor model[J].Chinese Medicinal Biotechnology,2008,3(2):116-120.
Authors:HU Ze-bin  LU Zhuo-zhuang  WU Zu-ze  WANG Li-sheng
Institution:(Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850, China)
Abstract:Objective To investigate the anti-tumor effect of a self-constructed recombinant oncolytic adenovirus Ad-CD80-TPE-GM in nude mice with Hep2 xenograft tumor. Methods Hep2 cells were subcutaneously injected into the right flank of nude mice to establish xenograft tumor models. The mice were divided into single and continuous treatment (one injection/d for 5 d) groups. In each group, the mice were subdivided into PBS, Ad-GFP, and Ad-CD80-TPE-GM subgroups. The PBS and Ad-GFP subgroups were set as the negative controls. In the single treatment group, the mice were killed on the 4th day after intra-tumoral injection, and then peripheral blood and the tumor were obtained. Limiting-dilution assay, BCA protein assay, and flow cytometry were used to detect the titer of adenovirus, concentration of total protein, and the positive expression rate of CD80. The expression levels of GM-CSF in peripheral blood and the tumor tissues were determined by ELISA. Then, the titer of adenovirus in the tumor tissues and the expression level of GM-CSF in total protein were calculated. In the continuous treatment group, the tumor was measured after the first intra-tumoral injection to calculate its volume. When the tumors in the controls were larger than 1 cm in diameter or ulcers were observed on their surface, the mice were killed to obtain the tumor tissues. Then, the relative rates tumor growth (T/C%) and inhibition were measured to investigate the anti-tumor effect of Ad-CD80-TPE-GM. Results In the single treatment group, the titer of adenovirus in Ad-CD80-TPE-GM subgroup was significantly higher than that in Ad-GFP subgroup 1.13 × 106 (1.40 × 105 - 7.25 × 107)IU/tumor vs. 1.17 × 102 (7.80 × 101 - 2.40 × 102)IU/tumor, P = 0.01], whereas, no adenovirus was detected in PBS subgroup; the expression level of GM-CSF in Ad-CD80-TPE-GM subgroup was (397.32 ± 179.67) pg/mg, which was significantly higher than that in PBS and Ad-GFP subgroups (4.02 ± 0.93) and (6.92 ± 2.79) pg/mg respectively, both P < 0.01]; the positive expression rate of CD80 in Ad-CD80-TPE-GM subgroup was 31.6% &;#61617; 9.0%, while CD80 could hardly be detected in the controls (both P < 0.001). In the continuous treatment group, the relative rate of tumor growth in Ad-CD80-TPE-GM subgroup was 31.8% (compared to PBS subgroup, P < 0.05) and 25.9% (compared to Ad-GFP subgroup, P < 0.01), respectively; and the relative rate of tumor inhibition was 73.4% and 77.9% (both P < 0.001). Conclusion Ad-CD80-TPE-GM can efficiently replicate and express target genes in Hep2 xenograft tumor models, showing strong anti-tumor effect.
Keywords:Adenoviridae  DNA  recombinant  Granulocyte-macrophage colony-stimulating factor  Antigens  CD80  Gene therapy
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