人内皮抑素转基因治疗B16黑色素瘤的实验研究

Anti-tumor effect through human endostatin gene transfer in mice bearing B16 melanoma

目的 研究瘤内直接注射携带人内皮抑素 (hEN)基因逆转录病毒包装的细胞株对体内B16黑色素瘤细胞生长的抑制作用。方法 修饰、鉴定hEN基因。构建含hEN基因的逆转录病毒载体pLNC hEN ,转染包装细胞株 ,筛选稳定转染该基因之PA317 pLNChEN细胞。建立小鼠B16黑色素瘤动物模型。瘤内直接注射PA317 pLNChEN细胞 ,分时间测量肿瘤体积 ,切取肿瘤 ,免疫组化检测肿瘤组织微血管密度 (MVD) ,TUNEL染色检测肿瘤组织中的凋亡细胞。结果 细胞接种后第 7～ 9天 ,全部小鼠均长出 2～ 3mm直径肿瘤。hEN基因转染后第 3,5 ,7,9天 ,转基因组肿瘤的平均体积 (mm3 )分别为 4 .6 7± 1.10、2 2 .2 5± 13.0 6、84 .17± 4 3.5和 15 5 .0 8± 81.1;空载体对照组分别为 136 .17± 30 .6 1、390 .17± 2 2 0 .4 7、10 2 1.6 7± 5 37.4 0和 2 92 0 .2 0± 2 2 0 .0 1,两组差异有显著性。转基因组和对照组的平均MVD分别为 8.0 0± 2 .2 8和 2 8.17± 5 .31,平均凋亡细胞数分别为 2 3.33± 3.83和 2 .33± 1.2 1,差异均有显著性。结论 瘤内注射携带hEN基因逆转录病毒包装的细胞 ,可抑制小鼠种植性B16黑色素瘤的微血管数目 ,促进肿瘤细胞凋亡 。

Objective To investigate the effects of direct intratumor injection of the packaged cells with retroviral vector carrying human endostatin (hEN) on the growth inhibition of B16 melanoma in C57/BL6 mice. Methods Retroviral vector, pLNC hEN, was constructed with modified and identified hEN gene. The cell line, PA317, was used to establish ecotropic virus producing cells by transfecting and packing with pLNC hEN. Then the cells were injected directly into the tumor in C57/BL6 mice bearing B16 melanoma established by intra cutaneous injection of B16 cell suspension. The tumor size was measured at different intervals to observe the antitumor effect. Micro vessel density (MVD) in the tumor tissue was evaluated by immunohistological examination to count the apoptotic cells by TUNEL staining. Results Tumor with diameter of 2 3 mm was observed in all mice after 7 9 days. The average tumor volume on D3, D5, D7 and D9 after gene transfection was 4.67±1.1, 22.25± 13.06, 84.17±43.5 and 155.08±81.1mm 3 in the gene therapy group but 136.17±30.61, 390.17± 220.47 , 1 021.67±537.4 and 2 920.2±220.01mm 3 in the control group, the difference of which was statistically significant. The average MVD in the gene therapy and control groups were 8±2.28 and 28.17±5.31 while the average apoptotic cell number in the two groups were 23.33±3.83 and 2.33±1.21, both of which were statistically significant. Conclusion The direct injection of packaged cells carrying hEN gene is able to inhibit the growth of micro blood vessels and promote tumor cell apoptosis, which ultimately inhibits the growth of B16 melanoma.