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目的探讨抗血管内皮生长因子(VEGF)发夹状核酶基因对白血病细胞裸鼠体内生长和肿瘤内血管生成的影响。方法采用脂质体介导的方法将抗 VEGF 发夹状核酶基因真核表达载体pcDNA-RZ 转染白血病细胞系 K562,G418抗性筛选获得阳性克隆;抽提基因组 DNA,用 PCR 方法验证核酶基因已转入 K562细胞;荧光定量 PCR 和免疫印迹反应检测白血病细胞中 VEGF mRNA 和蛋白表达量的改变;将白血病细胞皮下接种 BALB/c 裸鼠,观察转染细胞在裸鼠体内成瘤及生长情况;组织形态学和免疫组织化学法检测裸鼠移植瘤微血管密度(MVD)。结果抗 VEGF 发夹状核酶基因真核表达载体 pcDNA-RZ 转入白血病细胞系 K562(K562/RZ),G418筛选两周获得阳性克隆,PCR 检测证实核酶基因整合入白血病细胞基因组 DNA;与 K562及 K562/PC 细胞(转染空质粒的 K562细胞)相比,K562/RZ 细胞 VEGF mRNA 和蛋白的表达量明显降低。接种 K562、K562/PC 和 K562/RZ 细胞组小鼠肿瘤终体积分别为(3.21±0.89)cm~3,(3.42±1.01)cm~3,(1.71±0.94)cm~3;肿瘤重量分别为(4.43±0.87)g,(3.96±0.94)g,(2.24±0.56)g;瘤体内 MVD 分别为4.70±1.25,4.67±1.31和1.80±1.55。以上各组之间的差异均有统计学意义(P<0.01)。结论转导抗 VEGF 发夹状核酶基因能减少白血病细胞中 VEGF 的合成,细胞裸鼠致瘤能力明显减弱,瘤体内血管形成能力降低。
Objective To investigate the effect of anti-vascular endothelial growth factor (VEGF) hairpin ribozyme gene on the growth and tumor angiogenesis of leukemic cells in nude mice. Methods The positive clones were obtained by lipofectamine mediated transfection of pcDNA-RZ gene of anti-VEGF hairpin ribozyme gene into leukemia cell lines K562 and G418. The genomic DNA was extracted and verified by PCR Enzyme gene was transferred into K562 cells; Fluorescent quantitative PCR and Western blotting were used to detect the changes of VEGF mRNA and protein expression in leukemic cells; BALB / c nude mice were inoculated subcutaneously with leukemia cells, Growth conditions; Morphological and immunohistochemical detection of transplanted tumor microvessel density (MVD) in nude mice. RESULTS: The eukaryotic expression vector pcDNA-RZ of anti-VEGF hairpin ribozyme was transfected into K562 (K562 / RZ) leukemia cell line. Positive clones were screened by G418 for two weeks. PCR analysis confirmed the integration of ribozyme gene into genomic DNA of leukemia cells. The expression of VEGF mRNA and protein in K562 / RZ cells was significantly lower than that in K562 / K562 / K562 / PC cells (K562 cells transfected with empty plasmid). The tumor volume of mice inoculated with K562, K562 / PC and K562 / RZ cells were (3.21 ± 0.89) cm3, (3.42 ± 1.01) cm3 and (1.71 ± 0.94) cm3 respectively (4.43 ± 0.87) g, (3.96 ± 0.94) g and (2.24 ± 0.56) g, respectively; MVD in tumor was 4.70 ± 1.25, 4.67 ± 1.31 and 1.80 ± 1.55, respectively. The differences between the above groups were statistically significant (P <0.01). Conclusion The transduction of anti-VEGF hairpin ribozyme gene can reduce the synthesis of VEGF in leukemia cells. The tumorigenic ability of nude mice is obviously weakened and the ability of angiogenesis in the tumor is reduced.