丹参提取物单体对肿瘤坏死因子诱导内皮细胞组织因子表达的干预作用

Intervenient effect of 764-3 on TF expression induced by tumor necrosis factor-α in endothelial cells

研究丹参提取物单体 (76 4 3)对肿瘤坏死因子 α(tumornecrosisfactor α ,TNF α)诱导人血管内皮细胞 (humanvascularendothelialcell,HUVEC)组织因子 (tissuefactor,TF)基因表达的干预作用 ,及其对单个内皮细胞胞内游离钙([Ca2 + ]i)水平的影响 ,以探讨 76 4 3对心血管血栓栓塞性疾病防治的可能机制。进行人脐静脉原代内皮细胞、ECV30 4细胞株的培养。采用限制性内切酶法 ,构建含有人TF基因不同上游调控序列的荧光素酶报告基因质粒 ,经脂质体法转染内皮细胞 ,用TNF α及 76 4 3处理内皮细胞 ,测定细胞裂解物荧光素酶及 β半乳糖苷酶活性。以Fluo 3/AM为荧光指示剂 ,用激光扫描共聚焦显像系统观测 [Ca2 + ]i的改变。结果发现在TF基因上游序列 - 2 4 4 / +12 1bp存在下 ,TNFα可使转染内皮细胞荧光素酶表达量比对照组明显增加 ,76 4 3使这种增加有所降低。而 - 111/+12 1bp存在时 ,TNF α组与对照组荧光素酶表达量无明显差异 ,均比 - 2 4 4 / +12 1bp存在时明显降低 ,此时 ,76 4 3并未引起荧光素酶表达量明显改变。激光扫描共聚焦显像显示 ,TNF α可引起人内皮细胞 [Ca2 + ]i持续缓慢升高 ,加入 76 4 3后 [Ca2 + ]i迅速大幅度降低 ,逐渐恢复到基线水平。提示 76 4 3可抑制TNF

To investigate the intervenient effect of 764-3 on TFexpression and calcium ion([Ca 2+]i) induced by TNF-α in human vascular endothelial cells. We constructed two luciferase reporter gene containing different upstream sequences of TF gene. The two luciferase report genes, together with the intracontrol plasmid pSV-β-gal were respectively cotransfected into cultured human vascular endothelial cells. The relative luciferase activities were detected in ECV304 treated by TNF-α or/and 764-3. [Ca 2+] i in single HUVEC was observed with Laser-scanning confocal Microscope. TNF-α significantly increased the luciferase expression in the p-244/+121bp Luc transfected endothelial cells, the effect of TNF-α could be inhibited by 764-3. The luciferase expression in the p-111/+121bp Luc transfected endothelial cells was lower than that in the p-244/+121bp Luc transfected endothelial cells. TNF-α increase [Ca 2+] i in single HUVEC, but it was inhibted by 764-3. TF gene expression induced by TNF-α was inhibited by 764-3. [Ca 2+] iwas involved in it.