Chen Lin, Lü Wei, Wei Bo, Wang Ning, Li Tao
Department of General Surgery, General Hospital of Chinese PLA, Beijing 100853, China.
Zhonghua Yi Xue Za Zhi. 2009 Dec 1;89(44):3106-10.
To study the influence of inhibiting Delta-like ligand 4 (Dll4)/Notch signal transduction pathway upon the biological behavior of human umbilical vein endothelial cells (HUVEC).
Used rAAV vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the Dll4 (rAAV-Dll4-shRNA) to infect HUVEC. And an empty plasmid (rAAV-EGFP) was infected into the same cell line as control group. The stable transfection and expression of Dll4 mRNA in HUVEC were determined by semi-quantitative RT-PCR. The protein expression of Dll4 was examined by Western blotting. Distribution of cell cycle was assessed by flow cytometry. The cell growth was analyzed by MTT assay. HUVEC were separated by type I collagen and cultured in a three-dimensional culture system for tubule like structure (TLS) formation.
Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed the expression of Dll4 mRNA (0.636 +/- 0.082, 0.972 +/- 0.022 vs 0.948 +/- 0.046) and protein (0.632 +/- 0.052, 2.016 +/- 0.048 vs 1.946 +/- 0.066) were down-regulated in the stable cell (P = 0.024, 0.033). The rAAV vectors expressing an active small interfering RNA (siRNA) targeting the Dll4 effectively stimulated HUVEC cell growth and proliferation while empty plasmid had no such specific effect. The proliferation index of experimental group was (39.9 +/- 2.2)% versus untreated group (25.7 +/- 4.5)% (P = 0.036). TLS formation was significantly induced by rAAV vector. And the average length of TLS were more than those of control group (12.5 +/- 0.5, 8.7 +/- 7.7, 8.5 +/- 3.0, P = 0.028).
The inhibiting Dll4/Notch signal transduction pathway stimulates the proliferation of HUVEC and facilitates the angiogenesis. Interference with Dll4/Notch signaling may be particularly desirable in tumors with highly induced Dll4/Notch pathway.
研究抑制Delta样配体4（Dll4）/Notch信号转导通路对人脐静脉内皮细胞（HUVEC）生物学行为的影响。
用表达靶向Dll4的活性小干扰RNA（siRNA）的重组腺相关病毒载体（载体6）（rAAV-Dll4-shRNA）感染HUVEC。将空质粒（rAAV-EGFP）感染同一细胞系作为对照组。通过半定量RT-PCR测定HUVEC中Dll4 mRNA的稳定转染和表达。通过蛋白质印迹法检测Dll4的蛋白表达。通过流式细胞术评估细胞周期分布。通过MTT法分析细胞生长情况。用I型胶原分离HUVEC并在三维培养系统中培养以形成管状结构（TLS）。
与阴性对照细胞相比，半定量RT-PCR和蛋白质印迹法显示，稳定细胞中Dll4 mRNA（0.636±0.082，0.972±0.022对0.948±0.046）和蛋白（0.632±0.052，2.016±0.048对1.946±0.066）的表达下调（P = 0.024，0.033）。表达靶向Dll4的活性小干扰RNA（siRNA）的重组腺相关病毒载体有效刺激HUVEC细胞生长和增殖，而空质粒无此特异性作用。实验组的增殖指数为（39.9±2.2）%，而未处理组为（25.7±4.5）%（P = 0.036）。重组腺相关病毒载体显著诱导TLS形成。且TLS的平均长度大于对照组（12.5±0.5，8.7±7.7，8.5±3.0，P = 0.028）。
抑制Dll4/Notch信号转导通路可刺激HUVEC增殖并促进血管生成。在Dll4/Notch通路高度诱导的肿瘤中，干扰Dll4/Notch信号可能特别有用。
