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目的研究pU-VEGF-siRNA对恶性黑素瘤成瘤和凋亡的影响及其机制。方法构建针对血管内皮生长因子(VEGF)的发卡样siRNA真核表达载体pU-VEGF-siRNA,通过电穿孔法将构建重组体导入人恶性黑素瘤细胞系A375,并建立pU-VEGF-siRNA转染细胞荷瘤裸鼠模型,应用免疫组织化学方法检测荷瘤裸鼠肿瘤组织VEGF和血管内皮细胞特异性Ⅷ因子相关抗原(FⅧRAg)的表达,依据FⅧRAg的表达评价肿瘤微血管密度,末端脱氧核苷酸转移酶标记法(TUNEL)定量检测荷瘤裸鼠模型的肿瘤组织凋亡。结果体内实验表明,实验组成瘤率明显低于对照组,且其肿瘤生长速度也明显减慢(P<0.01)。实验组VEGF表达和肿瘤微血管密度明显低于对照组(P<0.01)。实验组可见大量凋亡细胞,对照组仅见少许凋亡细胞,实验组凋亡指数与对照组相比差异有统计学意义(P<0.01)。结论通过RNA干扰技术阻断VEGF的表达,在裸鼠体内可显著抑制恶性黑素瘤生长。
Objective To study the effect of pU-VEGF-siRNA on tumorigenesis and apoptosis of malignant melanoma and its mechanism. Methods The eukaryotic expression vector pU-VEGF-siRNA against VEGF was constructed and transfected into human malignant melanoma cell line A375 by electroporation, and the pU-VEGF-siRNA was constructed The tumor-bearing nude mice model was established. The expression of VEGF and vascular endothelial cell specific factor Ⅷ factor-related antigen (FⅧRAg) was detected by immunohistochemical method. The tumor microvessel density, terminal deoxynucleoside TUNEL method was used to detect tumor cell apoptosis in nude mice model. Results In vivo experiments showed that the rate of tumor formation in experimental group was significantly lower than that in control group, and the tumor growth rate also slowed down significantly (P <0.01). The expression of VEGF and the microvessel density in the experimental group were significantly lower than those in the control group (P <0.01). A large number of apoptotic cells were observed in the experimental group, while only a few apoptotic cells were seen in the control group. The apoptotic index in the experimental group was significantly different from that in the control group (P <0.01). Conclusion Blocking the expression of VEGF by RNA interference can significantly inhibit the growth of malignant melanoma in nude mice.