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

背景目前认为肿瘤坏死因子-α(TNF-α)可诱导人内皮细胞(HUVECs)组织因子基因的表达,而丹参提取物单体(764-3)对其干预作用如何?其机制有待于进一步探讨.目的研究764-3对TNF-α诱导HUVECs组织因子基因表达的干预作用,及单个内皮细胞内游离钙(Ca2+]1)水平的影响,以探讨764-3对防止心血管血栓栓塞性疾病的可能机制.设计随机对照的实验研究.单位协和医科大学基础医学研究所病理生理学系的实验室.材料实验于1998-05/1999-09在中国医学科学院,中国协和医科大学基础医学研究所病理生理学系实验室完成.脐带取自北京妇产医院.干预进行体外培养ECV304细胞株和人脐静脉内皮细胞,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(100 U/mL)及764-3(30 mg/L)处理细胞.主要观察指标测定细胞裂解物中荧光素酶及β半乳糖苷酶的活性.以Fluo-3/AM为荧光指示剂,用激光共聚焦显像系统观测Ca2+]i的改变.结果在TF基因上游序列-244/+121 bp存在下,TNF-α可使转染内皮细胞荧光素酶表达量较对照组明显增加,764-3使这种增加有所降低(P＜0.05).而-111/+121 bp存在时,TNF-α组较对照组荧光素酶表达量无明显差异,均比-244/+121 bp存在时明显降低,此时,764-3并未引起荧光素酶表达量明显改变.激光扫描共聚焦显像,TNF-α可引起人内皮细胞Ca2+]i持续缓慢升高,加入764-3后Ca2+]i迅速大幅度降低,逐渐恢复到基线水平.结论764-3可抑制TNF-α诱导的HUVECs TF基因表达的增强,这种作用依赖于该基因上游序列-244/+121 bp的存在,推测胞内游离钙离子可能参与此抑制作用.

Interventional effect of monomer extract of Radix Salviae Miltiorrhizae on tissue factor gene expression induced by tumor necrosis factor in endothelial cells

BACKGROUND: It is commonly thought that the expression of tissue factor (TF) gene in human umbilical vein endothelial cells (HUVECs) could be induced by tumor necrosis factor(TNF-α) . But the intervention effect of monomer extract of radix salviae miltiorrhizae(764-3) on TF expression in duced by TNF-α in endothelial cells has not been reported and the mechanism is still unclear.OBJECTIVE: To investigate the intervention effect of 764 - 3 on TF expression and calcium ion( Ca2+] i) induced by TNF-α in HUVECs so as to probe into the possible mechanism of 764 - 3 for preventing cardiovascular thromboembolic diseaseDESIGN: Randomized and controlled trial.SETTING: Laboratory of Department of Pathophysiology, Institute of Basic Medical Sciences, Peking Union Medical College MATERIALS: This study was conducted in the Department of Pathophysiology, Institute of Basic Medical Science, Peking Union Medical College,Chinese Academy of Medical Sciences from May 1998 to September 1999. Umbilical cord was chosen from Beijing Obstetrics and Gynecology Hospital.INTERVENTIONS: ECV304 cell strain and HUVECs were cultured in vitro. With gene recombination techniques, two luciferase reporter genes containing different length of human TF gene promoter were constructed. The two-luciferase reporter genes, together with the intracontrol plasmid pSV-3-gal were respectively cotransfected into cultured ECV304 and HUVECs.MAIN OUTCOME MEASURES: The activities of luciferase and βgalactosidase were detected in ECV304 and HUVECs treated by TNF-α or/and 764 -3. Taking Fluo- 3/AM as fluorescent indicator, Ca2+]i in single HUVEC was observed with laser-scanning confocal microscope.RESULTS: The luciferase expression in the p - 244/ + 121 bp luc transfected endothelial cells was significantly increased when the cells were exposed to 100 U/mL TNF-α. The induction of TNF-α could be inhibited by 764 -3 ( P ＜ 0.05). The luciferaseexpression in the p - 111/+ 121 bp Luc transfected endothelial cells was significantly lower than that in the p - 244/+ 121 bp ones and at the same time, 764 -3 did not cause the significantly change of the luciferase expression. Under laser-scanning confocal microscope, TNF-α increased Ca2 +] i in single HUVEC, but the effect was inhibited by 764 - 3.CONCLUSION: TF gene expression induced by TNF-α was inhibited by 764 - 3 in endothelial cells, which was dependent on the p-244/+ 121 bp,and Ca2+ ]; might be involved in it.