储存血小板经NF-κB信号通路引起人肺微血管内皮细胞损伤的实验研究  

Mechanism of platelet activation via NF-κB signaling pathway causing human pulmonary microvascular endothelial cell injury

作　　者：陈诚 张琦 夏荣 CHEN Cheng;ZHANG Qi;XIA Rong(Department of Transfusion,Affiliated Huashan Hospital of Fudan Universi-ty,Shanghai 200040,China)

机构地区：[1]复旦大学附属华山医院输血科,上海200040

出　　处：《中国输血杂志》2021年第11期1177-1180,共4页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金面上项目(82070197);中国输血协会威高科研基金(CSBT-WG-2017-11)。

摘　　要：目的探讨NF-κB信号通路在储存血小板对中性粒细胞介导的人肺微血管内皮细胞损伤过程中的作用,结果将有助于血小板介导输血相关急性肺损伤机制的进一步阐明。方法建立人肺微血管内皮细胞和中性粒细胞共培养体系,同时设立单纯共培养组和NF-κB抑制剂添加组,并同时添加储存3 d及5 d的血小板上清。分别检测NF-κB通路蛋白IκBα表达水平、细胞因子(IL-6、IL-8、IL-1β及TNF-α)表达水平、台盼蓝染色计算细胞死亡比率(死亡细胞数/细胞总数)及凋亡蛋白Caspase3表达水平。结果在单纯共培养组中,随着孵育时间延长,添加储存5 d血小板上清孵育后与对照相比,NF-κB通路蛋白IκBα表达水平(39 281.48±289.36 vs 112 67.68±407.27)明显降低(P<0.05);IL-6(169.85±32.49 vs 369.49±50.97)及IL-8(975.92±234.73 vs 1 854.28±373.85)表达水平存在差异(P<0.05),而IL-1β(31.43±2.84 vs 38.12±1.94)及TNF-α(48.24±2.68 vs 57.83±0.97)则无差异(P>0.05);细胞死亡比率(0.27±0.12 vs 3.33±0.31)、凋亡蛋白Caspase3表达水平(17 821.11±611.55 vs 42 064.42±542.86)均明显升高(P<0.05)。在抑制剂添加组中,添加储存5 d血小板上清孵育后与对照相比,NF-κB通路蛋白IκBα(41 928.17±386.26 vs 42 545.26±437.89)和IL-6(171.32±31.52 vs 177.37±31.43)、IL-8(969.53±229.58 vs 1 036.56±196.33)、IL-1β(31.42±2.71 vs 31.93±2.79)及TNF-α(48.33±2.51 vs 50.44±2.62)表达水平均无差异(P>0.05);细胞死亡比率(0.27±0.12 vs 1.26±0.11)仅添加储存5 d血小板上清孵育后与对照相比存在差异(P<0.05),其余则无差异(P>0.05)、凋亡蛋白Caspase3表达水平(10 502.77±457.55 vs 10 424.16±471.53)无差异(P>0.05)。结论 NF-κB可能参与了储存血小板调节炎症和凋亡进程,进而影响中性粒细胞介导的人肺微血管内皮细胞损伤的过程。Objective To explore the role of NF-κB signaling pathway in the process of platelet storage on neutrophil-mediated human pulmonary microvascular endothelial cell injury, in order to further clarify the mechanism of platelet-mediated transfusion-related acute lung injury. Methods A co-culture system of human lung microvascular endothelial cells and neutrophils was established. A simple co-culture group and NF-κB inhibitor addition group were set up simultaneously, and then the platelet supernatant stored for 3 and 5 days were added. The expression levels of NF-κB pathway protein IκBα and cytokines(IL-6, IL-8, IL-1β and TNF-α) were detected, the cell death ratio(the number of dead cells/total number of cells) and the expression level of apoptotic protein Caspase 3 were calculated by Trypan blue staining. Results In the simple co-culture group with 5-day stored platelet supernatant added, the expression level of NF-κB pathway protein IκBα decreased significantly as the incubation time prolonged(simple co-culture 39 281.48±289.36 vs control 11 267.68±407.27, P<0.05). Significant differences in the levels of IL-6(169.85±32.49 vs 369.49±50.97)and IL-8(975.92±234.73 vs 1 854.28±373.85)were noticed(P<0.05), but not in the levels of IL-1β(31.43±2.84 vs 38.12±1.94)and TNF-α(48.24±2.68 vs 57.83±0.97, P>0.05)when 5-day stored platelet supernatant was compared with 3-day stored one. The cell death rate(0.27±0.12 vs 3.33±0.31)and the expression level of apoptotic protein Caspase 3(17 821.11±611.55 vs 42 064.42±542.86)showed a significant increase(P<0.05). In the inhibitor addition group with 5-day stored platelet supernatant added, no significant difference was noticed in the expression level of NF-κB pathway protein IκBα(41 928.17±386.26 vs 42 545.26±437.89), IL-6(171.32±31.52 vs 177.37±31.43), IL-8(969.53±229.58 vs 1 036.56±196.33), IL-1β(31.42±2.71 vs 31.93±2.79)and TNF-α(48.33±2.51 vs 50.44±2.62, P>0.05)as compared with the controls. Significant difference in the cell death rate of

关 键 词：储存血小板 细胞因子 输血相关急性肺损伤 

分 类 号：R457.12[医药卫生—治疗学] R331.143[医药卫生—临床医学]