肿瘤坏死因子增强培养的血管内皮细胞与血小板粘附及其机制

Promoting effect of tumor necrosis factor on the adhesion of vascular endothelial cells to platelets and its mechanism

利用５１Ｃｒ－标记血小板检测其与内皮细胞粘附功能；ＥＬＩＳＡ方法检测血管内皮细胞表面玻璃连接蛋白受体（ＶｎＲ）表达改变；Ｆｕｒａ－２／Ａｍ负载测定群体细胞胞浆游离钙；Ｆｌｕｏ－３／Ａｍ负载测定单细胞内游离钙等方法，观察了肿瘤坏死因子（ＴＮＦ）对内皮细胞（ＥＣ）与血小板粘附功能的影响，发现ＴＮＦ－α（１００Ｕ／ｍｌ）可明显促进血小板（ＰＬ）与ＥＣ的粘附，ｃｐｍ值明显高于对照组（４１０．７±１７．６ｖｓ．２１９．７±１６．３ｎ＝６Ｐ＜０．０１）．用抗ＶｎＲ－β３亚单位的单抗（ＭｃＡｂ）可大部分阻断ＴＮＦ－α的促粘附作用，与未加单抗的ＴＮＦα组相比有显著差异，非ＭｃＡｂ组ｃｐｍ为４１０．５±１７．６，ＭｃＡｂ组为２８８．３±１３．８，（ｎ＝６，Ｐ＜０．０１），用ＥＬＩＳＡ法证实不同浓度ＴＮＦ－α（２００Ｕ／ｍｌ～１０００Ｕ／ｍｌ）可增加内皮细胞表面ＶｎＲ表达，以１０００Ｕ／ｍｌ组为最明显。同一浓度ＴＮＦ－α组，ＶｎＲ的表达呈现随作用时间延长而增加的趋势。此外，ＴＮＦ－α可增加ＥＣ［Ｃａ２＋］ｉ，群体细胞和单个细胞测定的结果一致。以上结果提示，ＴＮＦ－α可增加ＥＣ与血小板的粘附功能，其作用机制是以ＶｎＲ－（αｖβ３?

The effect of tumor necrosis facto-α(TNF-α) on the adhesion of ECs (endothelial cells)to platelets(PLs)and its mechanism was observed.The change of adhesion of ECs to PLs,the expression of vitronectin receptor(VnRe.g.integrin αVβ3)on the surface of ECs and the change of[Ca2+]i of mass-ECs and sigle EC were studied.It was found that cpm-value of TNF α-treated group was much higher than that of control group.(410.7±17.6 vs. 219.7±16.3 n=6 P<0.01).Anti-β3 monoclonal-antibody(McAb SZ-22)did block the main effect of TNFα-indrced adhesion of PLs to ECs.The cpm were 410.5±17.6 in TNFα+nonMcAb group and 288.3±13.8 in TNFα+McAb group (n=6 P<0.01).It was proved by ELISA method that different concentrations of TNFα(200U/ml-1000U/ml) could increase the expression of αVβ3 on the surface of EC and the most remarkable change was found in 1000U/ml group.In the same concentration group,the expression of VnR showed a up-regulated tendency with the stimulating time.In addition,TNFα indrced [Ca2+]i increase was consistant with both the mass-cell and single-cell.These results suggested that TNFα may strenthen the adhesive function of EC to PL,that the vitronection receptor (VnR,e.g,integrin αVβ3)involves in this vechanism as a main medium,via Ca^2+ as a second-messager.