血凝酶-海藻酸钙微球对人动脉血管内皮细胞炎症影响

Effect of haemocoagulase-loaded alginate-calcium microspheres on the inflammation of human arterial vascular endothelial cells

目的探讨血凝酶-海藻酸钙微球对人动脉血管内皮细胞(VECs)炎症的影响。方法将VECs细胞分为阴性对照组(正常培养基)、微球组(血凝酶-海藻酸钙微球浸提液)及阳性对照组(含0.64%苯酚的培养基或脂多糖100 ng/ml)。将血凝酶-海藻酸钙微球浸提液加入到VECs 24 h后,光学显微镜观察细胞的生长状态。采用CCK-8方法于24、48、72 h检测各组细胞活力。将微球浸提液加入到VECs 24 h后,采用荧光定量聚合酶链式反应和蛋白免疫印迹法检测白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的mRNA和蛋白表达情况。结果分组24 h后,微球组细胞数目和形态与阴性对照组相比无明显异常,而阳性对照组细胞明显皱缩,细胞形态明显异常,且存在大量的死亡细胞。与阴性对照组比较,微球组细胞给予刺激24、48、72 h后细胞活力均无明显变化(P>0.05);阳性对照组细胞活力在24、48、72 h明显降低(P<0.05)。与阴性对照组比较,微球组IL-6、TNF-α和IL-1βmRNA及蛋白表达水平无明显变化(P>0.05);阳性对照组IL-6、TNF-α和IL-1βmRNA及蛋白表达水平明显升高(P<0.05)。结论血凝酶-海藻酸钙微球对人VECs细胞活力和炎症无明显影响,与内皮细胞具有良好的细胞相容性。

Objective To investigate the effect of haemocoagulase-loaded alginate-calcium microspheres on the inflammation of human arterial vascular endothelial cells(VECs).Methods VECs cells were divided into the negative control group(normal medium),microsphere group(haemocoagulase-loaded alginate-calcium microspheres)and positive control group(medium containing 0.64%phenol or lipopolysaccharide 100 ng/ml).After adding the haemocoagulase-loaded alginate-calcium microspheres to VECs for 24 hours,the growth state of the cells was observed by optical microscope.The cell activity of each group was measured by CCK-8 method at 24,48 and 72 hours.After adding microsphere extracts to VECs for 24 hours,the mRNA and protein expressions of interleukin 6(IL-6),tumor necrosis factorα(TNF-α)and interleukin 1β(IL-1β)were detected by fluorescence quantitative polymerase chain reaction and protein Western blot.Results After 24 hours of grouping,the number and morphology of cells in the microsphere group were not significantly abnormal compared with those in the negative control group,while the cells in the positive control group were significantly shrivelled,the cell morphology was significantly abnormal,and there were a large number of dead cells.Compared with the negative control group,there was no significant change in cell activity in the microsphere group after 24,48 and 72 hours of cell stimulation(P>0.05).The cell activity of the positive control group was significantly reduced at 24,48 and 72 hours(P<0.05).Compared with the negative control group,the mRNA and protein levels of IL-6,TNF-αand IL-1βin the microsphere group showed no significant changes(P>0.05).The mRNA and protein levels of IL-6,TNF-αand IL-1βwere significantly increased in the positive control group(P<0.05).Conclusion The haemocoagulase-loaded alginate-calcium microspheres has no significant effect on the inflammation of human arterial vascular endothelial cells,but has good biocompatibility with endothelial cells.