目的 探讨新生儿脐血中性粒细胞(PMN)吞噬功能的变化及其影响因素.方法 收集2021年6—8月来自本院的健康成人血和脐血标本各13例,分离新鲜PMN,流式细胞仪检测PMN吞噬大肠杆菌的情况,ELISA法检测成人外周血和新生儿脐血血清中前列腺素E2(PGE2)的含量.应用不同浓度的PGE2(0-500 ng/ml)刺激PMN株HL-60细胞及PGE2(100 ng/ml)刺激新鲜分离的成人PMN 48小时后,流式细胞术观察HL-60细胞和PMN吞噬大肠杆菌的情况.结果 与成人PMN相比,新生儿PMN吞噬大肠杆菌百分率明显降低(P<0.01),但脐血PGE2的浓度较成人外周血明显升高(P<0.01);PGE2(100-500 ng/ml)刺激组HL-60细胞吞噬细菌百分率明显低于0 ng/ml组(P<0.01);成人PMN受PGE2(100 ng/ml)刺激后吞噬百分率明显降低(P<0.05).结论 新生儿脐血PMN吞噬功能降低,这可能是由于PGE2对PMN的抑制作用引起.

Abstract Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein‐coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50–1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL‐60 human neutrophils in a concentration‐dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL‐60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH‐6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

目的 探讨热休克蛋白70(heat shock protein 70,HSP70)对急性肺损伤(acute lung injury,ALI)小鼠发病的影响及其相关机制.方法 构建ALI模型小鼠,免疫印迹法检测肺组织中HSP70的表达情况,酶联免疫法检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中HSP70的分泌情况.ALI小鼠经鼻滴HSP70抗体(anti-HSP70)后,抽取BALF,检测其中肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白细胞介素6(interleukin 6,IL-6)、白细胞介素1β(interleukin 1β,IL-1β)和蛋白的含量,并计数中性粒细胞的数目;获取肺组织进行苏木精-伊红染色并计算肺湿/干重比.以LPS刺激小鼠肺泡上皮细胞株MLE-12细胞,酶联免疫法检测培养上清液中TNF-α和IL-1β的含量.不同浓度的HSP70(10～500 ng/mL)刺激小鼠肺泡上皮细胞(alveolar epithelial cells,AECs)后,流式细胞术检测AEC吞噬凋亡细胞的情况.结果 与正常对照组小鼠相比,ALI小鼠肺组织中HSP70的表达及BALF中HSP70的分泌均增加(均P<0.05).与ALI组小鼠相比,经anti-HSP70鼻滴治疗后的ALI小鼠,其BALF中TNF-α、IL-6、IL-1β和蛋白的含量均降低(均P<0.05),中性粒细胞数目减少(P<0.05);其肺组织中炎性细胞浸润减少,肺湿/干重比降低(P<0.05).与LPS刺激组相比,anti-HSP70+LPS组MLE-12细胞培养上清液中TNF-α和IL-1β含量均减少(均P<0.05).与未刺激组相比,10～500 ng/mL HSP70刺激组MLE-12细胞吞噬凋亡细胞百分率明显降低,其中以200 ng/mL HSP70刺激组降低最为明显(P<0.01);与未刺激组相比,200 ng/mL HSP70刺激的小鼠原代肺泡上皮细胞吞噬率降低(P<0.01).结论 HSP70促进ALI小鼠肺部炎症反应,这一效应可能与HSP70降低肺泡上皮细胞吞噬凋亡细胞的功能相关.

Objective To investigate the effect of insulin-like growth factor 1 (IGF-1) on the migration of alveolar epithelial cells (AECs) and its related mechanisms. Methods The MLE-12 cells (mouse AEC line) were stimulated by IGF-1 and sphingosine 1 phosphate (S1P) in the presence or absence of the PI3K inhibitor Wortmannin. Then, the cell migration was detected by the scratch test and the expression of p-Akt was detected by Western blot. With AECs stimulated by IGF-1, the secretion and expression of S1P were tested by ELISA and Western blot respectively. In the blocking experiment, the effect of IGF-1 on cell migration or p-Akt expression was detected by scratch test or Western blot after the interference of AEC S1P receptor 1 (S1PR1) or the action of S1PR1 blocking antibody. Results After 12 hours of IGF-1 stimulation, the expression of p-Akt in AECs increased and the migration of AECs accelerated. When blocking PI3K signal, the effect of IGF-1 on promoting AEC migration was partially eliminated. IGF-1 induced AECs to produce S1P, which accelerated AEC migration through S1PR1. The expression of p-Akt in AECs increased after S1P stimulation. When blocking the PI3K pathway, the ability of S1P to accelerate the migration of AECs was reduced. When S1PR1 in AECs was blocked or interfered, the effect of IGF-1 on accelerating AEC migration and promoting AEC p-Akt expression was partially reduced. Conclusion IGF-1 activates the PI3K pathway through S1P-S1PR1 signal to promote the migration of AECs.