Delta样分子4／Notch信号途径对人脐静脉内皮细胞生物学行为的影响  

Influence of Delta-like ligand 4/Notch signal transduction pathway upon the biological behavior of human umbilical vein endothelial cells

作　　者：陈凛[1] 吕伟[2] 卫勃[1] 王宁[1] 李涛[1] 

机构地区：[1]解放军总医院普通外科,北京100853 [2]第二炮兵总医院全军肝胆胃肠病中心

出　　处：《中华医学杂志》2009年第44期3106-3110,共5页National Medical Journal of China

基　　金：基金项目：国家自然科学基金（30872468）

摘　　要：目的特异性阻断Delta样分子4（D11）4／Notch信号途径，观察其对细胞生物学行为的影响。方法将自行构建的含短发夹样结构介导RNA干扰（aNAl）抑制D114基因表达的重组腺相关病毒（rAAV）载体（rAAV—DIM—shRNA）感染人脐静脉内皮细胞（HUVEC），以空病毒载体rAAV感染[HUVEC（-）组]及未转染细胞ttUVEC（HUVEC组）为对照。半定量逆转录（RT）-PCR，Western印迹分别检测HUVEC细胞D114mRNA和蛋白的改变；流式细胞技术分析细胞周期分布，MTT比色法检测细胞的生长抑制率，采用基质胶三维培养系统观测HUVEC体外血管形成能力的变化。结果HUVEC（＋）组和对照组[HUVEC组、HUVEC（-）组]D114mRNA表达与内对照GAPDH的比值分别为0．636_4-0．082、0．972-t-O．022和0．948±0．046（P=0．024），D114蛋白表达与内对照B-肌动蛋白的比值分别为0．6324-0．052、2．016±0．048和1．946±0．066（P=0．033）。rAAV-D114-shRNA转染HUVEC后，HUVEC（±）组及HUVEC组增殖指数分别为（39．9±2．2）％、（25．74-4．5）％（P=0．036）。HUVEC在基质胶中形成小管样结构72h时HUVEC（±）组与对照组[HUVEC组、HUVEC（-）组]细胞长度分别为（12．5±0．5，8．7±7．7，8．5±3．0，P=0．028）。结论D114／Notch信号途径对HUVEC生长、增殖、小管样结构形成能力发挥负性调控作用，特异性阻断D114／Notch信号途径将为进-步深入研究肿瘤新生血管生成、分化、成熟过程中提供新的思路和方法。Objective 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 ）. Methods Used rAAV vectors expressing an active small interfering RNA （siRNA） （vector 6） targeting the Dl14 （rAAV-Dllg-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 Dl14 mRNA in HUVEC were determined by semi-quantitative RT-PCR. The protein expression of DIM was examined by Western blotting. Distribution of cell cycle was assessed by flow cytometry. The cell growth was analyzed by MTr assay. HUVEC were separated by type I collagen and cultured in a three-dimensional culture system for tubule like structure （TLS） formation. Results Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed the expression of Dll4 mRNA （0. 636± O. 082, 0. 972 ± 0. 022 vs O. 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 DIM 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 ± O. 5, 8.7 ± 7.7, 8. 5 ± 3.0, P = 0. 028）. Conclusion The inhibiting Dll4/Notch signal transduction pathway stimulates the proliferation of HUVEC and facilitates the angiogenesis. Interference with D114/Notch signaling may be particularly desirable in tumors with highly induced Dll4/Notch pathway.

关 键 词：受体 NOTCH 细胞增殖 新生血管化 病理性 腺病毒科 Delta样分子 

分 类 号：R686[医药卫生—骨科学]