细胞培养基底刚度对人原代脐静脉内皮细胞增殖率及经登革病毒感染后释放 NO、ET-1的影响  

Effects of substrate stiffness on the proliferation of primary human umbilical vein endothelial cells and the release of NO and ET-1 during dengue virus infection

作　　者：余芳芳[1] 崔丽丽[1] 裴华[1] 马静[1] 左丽[1] 

机构地区：[1]贵阳医学院免疫学教研室,550004

出　　处：《中华微生物学和免疫学杂志》2015年第2期133-138,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（31260224）;贵州省教育厅“125”重大科技专项（黔教合重大专项字[2012]008号）

摘　　要：目的：探讨细胞培养基底刚度在登革病毒（ Dengue virus，DENV）感染人原代脐静脉内皮细胞（ HUVEC）中的影响。方法利用丙烯酰胺和双丙烯酰胺制备模拟正常人血管内皮细胞基底刚度的水凝胶[（0±4）kPa]，MTS法检测细胞的增殖，流式细胞仪检测细胞周期和凋亡，常规方法鉴定增殖登革病毒2型（DENV-2），DENV-2（MOI＝10）感染人原代脐静脉内皮细胞，MTS法比较种植在传统塑料基底与水凝胶上的HUVEC被DENV-2感染后对细胞增殖率的影响，并通过硝酸还原酶法检测两组细胞释放一氧化氮（NO）的水平以及双抗体夹心ELISA法检测内皮素-1（ET-1）的表达。结果自重弯曲法测得2％双丙烯酰胺和40％丙烯酰胺配比水凝胶的弹性模量为（3030±0．44） Pa；血管内皮细胞经登革病毒感染后其细胞增殖率与培养基底刚度呈正相关；两组不同刚度基底培养的细胞生长周期和细胞凋亡率无明显差异，但可引起NO和ET-1水平的变化。结论实验结果证实基底刚度可影响人原代HUVEC的增殖效率及释放的NO与ET-1浓度的变化，血管内皮受损后，其分泌合成的血管活性物质减少，参与了登革热的致病发生。同时水凝胶模型的建立为研究DENV感染的体外模型提供一定的新思路。Objective To investigate the effects of substrate stiffness on the proliferation of human umbilical vein endothelial cells （ HUVEC） during dengue virus infection.Methods Polyacrylamide gels were prepared for cell culture [（0±4） kPa].The proliferation of HUVEC cultured on substrates with differ-ent stiffness was determined by using 3-（4,5-diethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfo-phenyl）-2H-etrazolium,inner salt （ MTS） assay.The cycle and apoptosis of HUVEC were determined by flow cytometry analysis.Dengue virus serotype 2 （DENV-2） strains were propagated and identified by con-ventional assays.The HUVEC were infected with DENV-2 strains at a MOI of 10 and cultured on traditional plastic and hydrogel substrates, respectively.The levels of nitric oxide （NO） and endothelin-1 （ET-1） were detected by nitric acid reductase assay and double antibody sandwich ELISA.Results Young′s modulus E value of the hydrogels was （3030 ±0.44） Pa.The proliferation of HUVEC and the expression of NO and ET-1 were enhanced along the increased substrate stiffness.However, no significant differences with the cell cycle and apoptosis were observed between cells cultured on different substrates.Conclusion The stiffness of substrates affected not only the proliferation of HUVEC, but also the release of cytokines during DENV-2 infection.The development of dengue fever was associated with the decreased secretion of vascular active substances as a result of blood vessel injury.The establishment of hydrogel substrates as the model of vascu-lar basement membranes might provide a new way for the in vitro investigation of the pathogenesis of DENV infection.

关 键 词：基底刚度 登革病毒 人原代脐静脉内皮细胞 增殖 细胞因子 

分 类 号：R512.8[医药卫生—内科学]