抗内皮细胞抗体介导的内皮细胞损伤机制

目的探讨抗内皮细胞抗体（antiendothelial cell antibody，AECA）对内皮细胞凋亡的影响及重组α-烯醇化酶对内皮细胞凋亡的阻断作用。方法以EA．hy926细胞（内皮细胞永生细胞）提取物蛋白为抗原，采用免疫印迹法检测并筛选47000-AECA阳性患者血清，用蛋白G亲和层析法纯化IgG，在体外诱导内皮细胞凋亡，观察重组α-烯醇化酶对凋亡的阻断作用。结果含47000-AECA IgG在体外可诱导内皮细胞凋亡，荧光染色可见明显的核形态变化及典型的凋亡小体；含47000-AECA系统性红斑狼疮患者的IgG作用于EA．hy926细胞24、48和72小时后，凋亡率均明显高于相同剂量正常人kG作用EA．hy926细胞的凋亡率；而经重组α-烯醇化酶预处理后EA．hy926细胞凋亡率则明显降低。含47000-AECA IgG作用于EA．hy926细胞8小时后，AnnexinV^＋细胞数明显增加，并随IgG作用时间和浓度的增加而升高；而经重组α-烯醇化酶预处理的含47000-AECA IgG作用于EA．hy926细胞后，AnnexinV^＋细胞有所减少。结论AECA在体外可诱导内皮细胞凋亡，引起内皮细胞损伤；重组α-烯醇化酶可部分阻断47000-AECA体外诱导内皮细胞凋亡的作用，α-烯醇化酶是AECA识别的自身抗原之一。

Mechanism of Endothelial Cell Injury Mediated by Antiendothelial Cell Antibody

Objective To determine whether antiendothelial cell antibody （AECA） induces apoptosis in endo- thelial ceils （ECs） and whether α-enolase is a target antigen. Methods Patient sera positive for 47 000- AECA were assayed by immunoblotting. IgG fractions of control sera and patient sera were purified over a protein G-Sepharose column. Purified IgG at different concentration was preincubated or was not preincu- bated with α-enolase and added in the culture medium to stimulate the EA.hy926 Cells. Apoptosis was evaluated using a fluorescence microscope and a flow cytometer by measuring FITC-conjugated annexin V staining and propidium iodide （PI）. Results In vitro incubation of ECs with 47 000-AECA containing IgG induced apoptosis in a time- and dose-dependent manner, as determined by Hoechst 33 342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Apoptosis was partly inhibited by preincubation of the ECs with recombinant α-enolase. Conclusion AECA may cause vascular dysfunction by inducing apoptosis. Apoptosis is partly inhibited by preincubation of the ECs with recombinant α-enolase. Human α enolase is one of the antigens recognized by AECA.