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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 ,抑制肿瘤生长
Objective To study the inhibitory effects of direct injection of retrovirus packaging cells carrying human endostatin (hEN) gene on the growth of B16 melanoma cells in vivo. Method modification, identification of hEN gene. The hEN gene-containing retrovirus vector pLNC hEN was constructed and transfected into packaging cell lines to screen PA317 pLNChEN cells stably transfected with this gene. Mouse B16 melanoma animal model was established. The tumor cells were directly injected with PA317 pLNChEN cells in a time-dependent manner. The tumors were excised, the MVDs were detected by immunohistochemistry, and the apoptotic cells were detected by TUNEL staining. Results All the mice grew 2 ~ 3mm diameter tumors on the 7th to 9th days after inoculation. The mean tumor volume (mm3) of the transgene group on the 3rd, 5th, 7th and 9th day after hEN gene transfection were respectively 4.67 ± 1.10, 22.5 ± 13.0 6, 84.17 ± 4 3.5 and 15 5 .0 8 ± 81.1; empty vector control group were 136.17 ± 30.61 1,390.17 ± 2 2 0.4 4.7 10 2 1.6 7 ± 5 37.4 0 and 2 92 0 .2 ± 2 2 0 .0 1, the difference between the two groups was significant. The mean MVD of the transgenic and control groups were 8.0 ± 2.28 and 2 8.17 ± 5.31, respectively, and the average number of apoptotic cells was 23.33 ± 3.83 and 2.33 ± 1.2 1, respectively. The differences were significant. Conclusion Intrathecal injection of hEN-retrovirus-encapsulated cells can inhibit the number of microvasculature of implanted B16 melanoma in mice, promote tumor cell apoptosis and inhibit tumor growth