TNF-α、IL-1β和LPS诱导人脐静脉内皮细胞增殖和凋亡的研究

Effect of TNF-α, IL-1β and LPS on proliferation and apoptosis of human umbilical vein endothelial cells

摘要:

摘要：目的  体外观察眼表烧伤后早期的炎症因子，如肿瘤坏死因子-α（TNF-α）、白介素1β（IL-1β），以及晚期的炎症因子脂多糖（LPS）对体外培养的人脐静脉内皮细胞（HUVEC）增殖和凋亡的影响，并为下一步探讨眼表烧伤后不同时期炎症致内皮损伤的机制提供理论基础。方法  本实验随机分为4组：正常组、TNF-α组、IL-1β组和LPS组。采用活细胞计数法试剂盒（CCK-8）法测定细胞的增殖活性，台盼蓝排除染色法检测细胞的增殖数量，吖啶橙/溴化乙锭（AO-EB）染色法检测细胞的凋亡，以及采用流式细胞仪检测细胞的凋亡率。结果  ①CCK-8法检测结果显示，正常组细胞的增殖活性增高，其余3组细胞的增殖活性下降，且LPS组细胞的增殖活性下降的趋势大于IL-1β组和TNF-α组，IL-1β组细胞增殖活性下降的趋势大于TNF-α组；②台盼蓝排除染色法的定量检测结果趋势类似于CCK-8法检测结果；③AO-EB染色结果显示，正常组细胞被吖啶橙（AO）染成绿色或黄绿色，细胞形态和结构正常，未见细胞核着橘红色荧光的凋亡细胞或死亡细胞，而其余3组均可见细胞核着橘红色荧光的凋亡细胞或死亡细胞。流式细胞仪检测4组细胞的凋亡率，同样TNF-α组、IL-1β组和LPS组细胞的凋亡率均高于正常组细胞，且LPS组细胞的凋亡率大于IL-1β组；IL-1β组细胞的凋亡率大于TNF-α组，差异有统计学意义（P <0.05）。结论  早、晚期的炎症介质TNF-α、IL-1β及LPS均可降低HUVEC的增殖活性，增加其凋亡率，但诱导的程度不同，这可能与不同炎症介质激活的主要分子信号通路不同有关。

Abstract:

 Abstract: Objective To investigate the effect of TNF-α, IL-1β and LPS on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro, so as to further study the mechanism of endo-thelial cell injury induced by inflammatory cytokines in different period after eye burn. Methods The HUVECs were randomly divided into control group, TNF-α group, IL-1β group and LPS group. Cell proliferation acti-vity was assessed by CCK-8. The number of proliferative cells was detected by trypan blue exclusion staining. The apoptosis was assessed by AO-EB staining. The apoptosis rate was detected by flow cytometry. Results Compared to the control group, the cell proliferation activity in the other 3 groups were markedly decreased in a  downward trend from TNF-α group, IL-1β group to LSP group. Meanwhile, the results of proliferative cell counting were also similar with CCK-8 results. The cell structures in the control group were normal, and apoptotic cells were observed in the other 3 groups. Compared with the control group, the apoptosis rate of HUVECs in the TNF-α, IL-1β and LPS groups was significantly increased; and the cell apoptosis rate of the LPS group was markedly higher than that of the TNF-α and IL-1β groups, and the cell apoptosis rate of the IL-1β group was markedly higher than that of the TNF-α group (P < 0.05). Conclusions The inflammatory cytokines such as TNF-α, IL-1β and LPS in early and late stages after burn can significantly decrease proliferative activity of HUVECs and increase the apoptosis rate in different degrees. This may be related to different signaling pathways activated by different inflammatory mediators.