Tenomodulin蛋白对血管内皮细胞增生的抑制作用

目的研究tenomodulin（TeM）蛋白对血管内皮生长因子（VEGF）诱导的人视网膜血管内皮细胞（RECs）和人脐静脉内皮细胞（UVECs）增生及其对体外血管样结构形成的作用。方法将传代至3～6代的人RECs和人UVECs生长至融合时用含质量分数0.5%胎牛血清（FBS）的培养基孵箱中培养6h,加入不同质量浓度的VEGF或VEGF＋TeM混合物,继续培养12h,ELISA法检测TeM对VEGF诱导的血管内皮细胞增生的影响。将含10%FBS、1%FBSDMEM培养基的人RECs和人UVECs悬液分别接种于预先放置基质胶的24孔培养板表面,含1%FBS培养基的细胞培养板内分别加入VEGF（100μg/L）或VEGF＋TeM混合物37℃继续培养6h,共焦显微镜下观察,图像处理分析软件量化分析TeM蛋白对毛细血管样结构形成的作用。结果随着VEGF质量浓度的增加,血管内皮细胞DNA合成增加,其吸光度（A）值呈明显上升曲线,而添加TeM组的A值比VEGF组降低,差异有统计学意义（P〈0.01）。含10%FBS和1%FBS培养基的血管内皮细胞在基质胶表面形成毛细血管样结构,并连接成网状;在基质胶表面同时添加VEGF的血管内皮细胞,其毛细血管样结构明显增多,长度明显变长。添加TeM组的血管内皮细胞和其毛细血管样结构破坏,每个视野内血管内皮细胞毛细血管样结构显著破坏,管形细胞总长度明显变短。结论 TeM蛋白对血管内皮细胞的增生及体外血管样结构的形成有明显抑制作用。

Inhibitory effect of tenomodulin protein on proliferation of vascular endothelial cells

Background Vascular endothelial growth factor （VEGF） participates in retinal neovascular formation.Researches showed tenomodulin（TeM） can inhibit the new vessel of tumor tissue.Objective This study was to explore the effect of tenomodulin protein on cell proliferation and capillary-like morphogenesis of vascular endothelial cells under the stimulation of vascular endothelial growth factor （VEGF） in vitro.Methods Human retinal vascular endothelial cells （RECs） and human umbilical vein endothelial cells （UVECs） were cultured and passaged in HuMedia-EG2 and DMEM containing 10% fetal bovine serum.The cells from generation 3 through 6 were seeded in a 96-well plate to grow to subconfluence.The cells were then starved in DMEM containing 0.5%FBS for 6 hours and stimulated with VEGF or the mixture of VEGF and TeM protein at the indicated concentration for another 12 hours.BrdU ELISA chemiluminescence was used for cell proliferation according to the manufacturer’s protocol.Human RECs and human UVECs starved in 1%FBS containing culture medium were harvested with trypsin/EDTA and were seeded on polymerized Matrigel in the presence of VEGF or the mixture of VEGF and TeM at the indicated concentrations.Cells were also seeded in 10%FBS containing culture medium as a positive control.The plate was incubated at 37 ℃ for 6 hours and then photographed.Capillary-like morphogenesis of human RECs and human UVECs was quantitatively evaluated,and total lengths of tube-like structures per field were measured in a masked way.Results Cultured human RECs and confluent human UVECs showed the cobblestone-like arrangement in 4-7 days under the confocal microscope.The A value of human RECs and human UVECs in only VEGF group was elevated and that in VEGF＋TeM group was declined.The capillary-like structure formed by human UVECs and human RECs under the stimulation of VEGF were intact in 10%FBS culture group,1%FBS culture group and 1%FBS＋100 μg/L VEGF group;however,the capillary-like structure formation completely impaired in 1%FBS＋100 μg/L VEGF ＋1 mg/L TeM group.The total length of the capillary-like structures per field was significantly shorter in the medium with TeM than that of without TeM （P0.01）.Conclusion TeM shows a dominantly inhibitory effect on proliferation of human RECs and human UVECs and the capillary-like morphogenesis of vascular endothelial cells in vitro.