抗中性粒细胞胞浆抗体相关抗原诱导人胎肾内皮细胞凋亡的研究

Apoptosis of human fetal glomerular endothelial cell induced by neutrophil proteinase 3 from ANCA positive children with SLE

目的 探讨抗中性粒细胞胞浆抗体 (ANCA)相关抗原蛋白酶 3(PR 3)对体外培养人胎儿肾小球内皮细胞凋亡的影响及诱导机制。方法 采用ANCA阳性系统性红斑狼疮 (SLE)患儿外周血中性粒细胞分离纯化其细胞质中PR 3 ,并用该蛋白酶诱导培养人胎肾内皮细胞发生凋亡。观察不同剂量PR 3于不同时间点对内皮细胞存活情况及其形态学变化的影响。用碘化丙啶染色DNA以评价凋亡的发生率 ,观察核染色质固缩及DNA片段的形成。用琼脂糖凝胶电泳确定凋亡小体的形成和凋亡梯形。结果 ANCA阳性SLE患儿中性粒细胞胞浆蛋白酶PR 3以剂量、时间依赖性形式诱导人胎肾内皮细胞发生凋亡 [PR 3 10 μg·ml-1·2 4h-1细胞凋亡率为 (75 5± 2 6 5 ) % ],与阴性对照组 [(1 8± 1 2 ) % ]比较差异有显著性 (P <0 0 1)。凋亡的内皮细胞与培养基分离、悬浮、呈碘化丙啶强染的大小不等的碎片 ,核染色体固缩。DNA电泳提示凋亡核小体形成 ,出现凋亡梯形。结论 中性粒细胞胞浆蛋白酶PR 3可诱导胎肾内皮细胞凋亡 。

Objective To investigate whether the neutrophil serine proteinase 3 (PR 3),a specific autoantigen of C ANCA,could induce human fetal glomerular endothelial cell apoptosis in vitro .Methods PR 3 was isolated from polymorphonuclear leukocytes obtained from C ANCA positive children with SLE.The cultured glomerular endothelial cells from human fetal kidneys were treated with PR 3 and apoptosis was assessed by different methods including the propidium iodide staining,DNA fragmentation analysis,and morphology observation.Results Percentage of apoptotic cells significantly increased ( P <0 01) with 10 μg/ml PR 3 at hour 24 after treatment.Values at this time point were (75 5±26 5)% for PR 3 group and (1 8±1 2)% for negative control,demonstrating an increase of apoptosis in a dose and time dependent manner with the highest values achieved at hour 24.The apoptosis features appeared as nuclei condensation and DNA fragmentation,and typical DNA laddering was apparent in cells treated with PR 3 and in positive control cells treated with TNF α.Conclusion The results suggest that PR 3 may directly induce glomerular endothelial cell apoptosis in vitro .This pathogenic role of PR 3 may be an important machanism of injury in anti neutrophil cytoplasmic antibody (ANCA) associated vasculitic diseases in children.