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目的探索C5a是否与LPS、毒源性大肠杆菌O157产生的外毒素—V毒素(verotoxin)具有相似的可以导致血管内皮细胞(VEC)凋亡的作用。方法流式细胞仪检测细胞凋亡发生率,首先探讨rhC5a刺激的质量浓度,分别为0.2μg/mL、0.5μg/mL、1μg/mL和1.5μg/mL刺激12 h检测VEC凋亡发生率;在rhC5a质量浓度均为1μg/mL条件下,时间分别为2 h、6 h、12 h、18 h和24 h,检测VEC凋亡发生率。Western blot方法检测凋亡相关蛋白。结果在rhC5a质量浓度为0.2μg/mL、0.5μg/mL、1μg/mL和1.5μg/mL刺激12 h诱导的凋亡发生率分别为4.58%、7.87%、17.94%和19.03%。在确定rhC5a质量浓度1μg/mL的条件下,VEC发生凋亡呈时间依赖关系,在2 h、6 h、12 h、18 h和24 h的时相,细胞凋亡发生率分别为4.58%、12.78%、18.12%、19.08%和19.96%。rhC5a刺激VEC细胞,calpain-2蛋白表达呈时间依赖性,而calpain-1蛋白的表达为浓度依赖性。结论C5a可以导致VEC细胞发生凋亡,calpain参与了这类细胞的凋亡进程。
Aim To investigate whether C5a has similar effects on apoptosis of vascular endothelial cells (VECs) as exotoxin-V toxin produced by LPS and virulent Escherichia coli O157. Methods Flow cytometry was used to detect the incidence of apoptosis. First, the concentration of rhC5a stimulated by rhC5a was measured and the incidence of VEC apoptosis was detected at the concentration of 0.2μg / mL, 0.5μg / mL, 1μg / mL and 1.5μg / mL, respectively. The incidence of VEC apoptosis was detected at 2, 6, 12, 18, and 24 h after the rhC5a concentration was 1 μg / mL. Apoptosis-related proteins were detected by Western blot. Results The apoptotic rates induced by rhC5a at concentrations of 0.2μg / mL, 0.5μg / mL, 1μg / mL and 1.5μg / mL for 12 h were 4.58%, 7.87%, 17.94% and 19.03%, respectively. The apoptotic rate of VEC in time-dependent manner was determined at the concentration of 1μg / mL rhC5a, and the apoptotic rate was 4.58% at 2 h, 6 h, 12 h, 18 h and 24 h respectively, 12.78%, 18.12%, 19.08% and 19.96%. The expression of calpain-2 protein was time-dependent in rhC5a-stimulated VEC cells, while the expression of calpain-1 protein was concentration-dependent. Conclusion C5a can induce apoptosis in VEC cells and calpain is involved in the apoptosis of these cells.