人血管生成素-1对血管内皮细胞增殖及凋亡影响的研究

The Effect of Human Angiopoietin-1 on the Proliferation and Apoptosis of Human Umbilical Vein Endothelial Cell

目的:探讨人血管生成素-1(angiopoietin—1,Ang-1)对人脐静脉内皮细胞(human umbilican vein endothelia cell,HU-VEC)增殖和凋亡的影响,以进一步研究其生物学作用及其在肿瘤发生中的作用机制。方法:构建.pcDNA3.1-V5-HisC-Ang-1真核表达载体,并瞬时转染293细胞;取新生儿脐带经胶原酶消化等方法分离培养HUVEC;分别通过MTT比色和细胞计数法,分析Ang-1瞬时转染上清对HUVEC增殖的影响;通过流式细胞仪分析在血浆饥饿实验条件下,Ang-1对HUVEC凋亡的影响。结果:成功克隆了Ang-1基因,构建了其正义真核表达载体,并在293细胞中瞬时表达;成功进行了HUVEC的原代分离及传代培养;MTT法检测HUVEC增殖结果:只加培养液组、加空载体转染上清组、加Ang-1转染上清组HUVEC OD490值分别为0.36±0.11,0.40±0.03,0.68±0.10(P<0.05);细胞计数法检测结果依次为:(10.13±2.06)×104,(8.7±1.73)×104,(15.03±1.98)×104(P<0.05)。流式细胞仪检测HUVEC凋亡率依次为:21％,19％和6％。结论:Ang-1能显著促进血管内皮细胞体外增殖,抑制血浆饥饿时HUVEC的凋亡。

Objective: To investigate the effect of human Angiopoietin-1 (Ang-1) on the proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods: The recombinant eukaryotic expression vector pcDNA3. 1-V5-HisC-Angl was constructed and was used to transiently transfecte to 293 cells. HUVEC was seperated from new born fetel cord. The supernatants from transfected 293 cells were harvested, and then added to HUVEC culture. The proliferation and apoptosis of HUVEC was measured by MTT colorimetry assay analysis, cell counting and flow cytometry, respectively. Results: Proliferation of HUVEC cells cultured with supurnatants of Ang-1 transfected group was higher than those untreated and mock transfected group, results of which were 0. 68 ±0. 10, 0. 36 ± 0. 11, 0. 40 ± 0. 03 in the MTT assays; and were ( 15. 03 ± 1. 98) × 104 , ( 10. 13 ±2. 06) × 104 and (8. 7 ± 1. 73) × 104 respectively in the cell counts. The ap-optotic pereentage of HUVEC cells culturel with supernatants from Ang-1 transfected group was lower than those of untreated or mock transfeeted group, results of which were 6% ,21% and 19% respectively in FACS analysis. Conclusions: Human Ang-1 can significacetly pronaete stimulate the proliferation and reduce the apoptosis of endothelial cells.