人脐静脉血管平滑肌促内皮细胞组织因子表达的体外实验研究

IN VITRO STUDY OF TISSUE FACTOR EXPRESSION IN ENDOTHELIAL CELIS STIMULATED BY HUMAN UMBILICAL VEIN SMOOTH MUSCLE CELLS

目的 研究血管平滑肌细胞对血管内皮细胞组织因子表达的影响并探讨其临床意义。方法 用贴块法培养人脐静脉平滑肌细胞 ;酶消化法培养人脐静脉内皮细胞 ;用培养平滑肌细胞条件培养液 (SMC CM)刺激培养的内皮细胞 ,一步凝固法检测内皮细胞组织因子的活性 ;Northernblot检测内皮细胞组织因子的mRNA表达 ;并用酶联免疫吸附试验检测SMC CM中IL 1α、IL 1β、TNF α和VEGF的含量。结果 SMC CM使内皮细胞组织因子活性呈剂量依赖性增强 ,作用 6h增至最高 ,最高增强约 38倍 ;SMC CM使内皮细胞组织因子mRNA表达显著增强 ;SMC CM中的组织因子诱导剂不耐热 ,且并非IL 1α、IL 1β、TNF α和VEGF等已知的组织因子诱导剂。结论 血管平滑肌细胞能促进血管内皮细胞组织因子的表达 ,提示体内增生的平滑肌细胞 ,如动脉再狭窄新内膜中的平滑肌细胞可能诱导局部血管内皮细胞活化及表达组织因子 ,在局部血栓形成中起一定作用。

Objective To investigate the influence of vascular smooth muscle cells on the tissue factor expression in vascular endothelial cells. Method Human umbilical vein smooth muscle cells (HUVSMC) were cultured by a substrate-attached explantmethod. Human umbilical vein endothelial cells (HUVEC) were cultured byatrypsin digestion method.Tissue factor (TF)activity was measured in the lysates of HUVEC by using a single step clotting assay. A specific mRNA expression was determined by Northern blotting. IL-1α,IL-1β,TNF-α and VEGFwere measured in the conditioned medium of HUVSMC(SMC-CM)by a specific enzyme-linked immunosorbent assay (ELISA). Result The conditioned medium from cultured HUVSMC inducedTF activity in HUVEC in a dose- and time-dependent fashion. TF activity peaked in HUVEC after 6 h incubation with SMC-CM, which was approximately a 38-fold increase in TF activity over the control level. This TF induction was also seen in the level of specific TF mRNA as evidenced by Northern blotting. The TF-inducing activity was heat-labile and was found to be distinct from the known inducers of TF expression in endothelial cells such as interleukin-1, tumor necrosis factor-α and vascular endothelial growth factor.Conclusion Vascular smooth muscle cells could induce TF expression in vascular endothelial cells. TF inducing factor released by proliferating smooth muscle cells in vivo, such as smooth muscle cells in neo-intima formation, could contribute to the activation of endothelial cells and the expression of TF, which might play a role in local thrombosis.