去甲斑蝥素对人真皮淋巴管内皮细胞体外三维培养淋巴管生成的影响及其机制

Inhibitory effect of norcantharidin on human dermal lymphatic endothelial cell in three-dimensional culture

目的探讨去甲斑蝥素(NCTD)对人真皮淋巴管内皮细胞(HDLEC)体外三维培养淋巴管生成的影响及机制。方法建立HDLEC体外培养淋巴管生成模型,随机分为NCTD组(加1/3 IC_(50)NCTD 40 nM)与空白对照组,作用24 h,观察两组淋巴管生成的形态变化;并采用免疫细胞化学、Western blot试验和qPCR技术测定比较两组淋巴管生成因子(VEGF—C、VEGF—D和VEGFR—3)蛋白和基因表达。结果 NCTD组的HDLEC细胞形态发生改变,很少形成管状结构,时间延长后细胞出现变圆、离壁、浮起、死亡;且NTCD组淋巴管生成(10.9±2.3)个,明显少于空白对照组(68.4±5.2)个(P<0.01)。免疫细胞化学染色、Western blot试验结果显示,NCTD对HDLEC三维培养淋巴管生成中的VEGF—C和VEGF—D的蛋白表达下调(P<0.01)。qPCR检测显示,NCTD对VEGF—C、VEGF—D和VEGFR—3的基因表达明显降低(P<0.01)。结论 NCTD对通过下调淋巴管生成因子VEGF—C、VEGF—D的蛋白和基因表达,产生抗淋巴管生成的作用。

Objective To study the effect of norcantharidin （NCTD） on human dermal lymphatic epithelial cell （HDLEC） in three-dimensional culture and related molecular mechanisms. Methods Three dimensional model of lymphangiogenesis with HDLEC was established to mimic lymphatic capillary-like structures in vitro. The three-dimensional cultured HDLEC were randomly assigned to control group（no NCTD） and NCTD group（cultured with 40 nm NCTD） for 24 h. The morphological changes were observed by inverted microscope, the expressions of VEGF-C, VEGF-D, VEGFR-3 protein and MicroRNA in three- dimensional cultured HDLEC were measured by immunohistochemistry, western-blot and qPCR. Results The lymphangio-genesis model in vitro was established successfully by three-dimensional culture of HDLEC. The inhibitory effects of NCTD on lymphangiogenesis in vitro were observed. Compared to control group, the expression of VEGF-C and VEGF-D proteins in NCTD group was significantly down-regulated as showed by immunohistochemistry and Western-blot （P〈0.01）, The MicroRNA expression of VEGF-C, VEGF-D and VEGFR-3 in NCTD group was significantly decreased as shown by qPCR （P〈0.01）. Conclusion Norcantharidin inhibits lymphangiogenesis by down-regulating the expression of VEGF-C and VEGF-D, suggesting that it might be an effective agent for targeting anti-lymphangiogenesis.