摘要
目的观察牙髓干细胞(DPSC)来源的外泌体对脂多糖(LPS)诱导大鼠肺泡巨噬细胞急性肺损伤(ALI)模型的作用及机制。方法 DPSC培养至第6代后,换无血清培养基处理24 h,收集上清液,采用超速离心法分离外泌体。取对数生长期的大鼠肺泡巨噬细胞(PAM)NR8383接种于12孔板,分别用1μg/ml LPS或联合外泌体处理细胞。在细胞处理前(0 h)和处理后6、12、24 h收集细胞上清液,通过酶联免疫吸附实验(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和IL-6。裂解提取细胞蛋白,采用Western印迹法测定细胞外调节蛋白激酶(p44/42)、转录因子NF-κB(p65)、IκBα蛋白的表达量及其磷酸化水平。结果与对照组比较,LPS处理组TNF-α、IL-1β和IL-6水平显著增高(P<0.05),提示LPS诱导ALI细胞造模成功。与LPS处理组相比,高剂量外泌体处理后,TNF-α和IL-1β表达量明显降低(P<0.05);IL-6在低剂量和高剂量的外泌体处理后均有明显降低(P<0.05)。DPSC来源的外泌体能降低转录因子NF-κB(p65)、IκBα以及p44/42的磷酸化水平。结论 DPSC来源的外泌体对LPS诱导的ALI有保护作用,其机制可能与抑制MAPK和NF-κB信号通路的激活有关。
Objective To investigate the protective role and underlying mechanism of human dental pulp stem cells（ DPSCs）-derived exosomes against lipopolysaccharide（ LPS） induced acute lung injury（ ALI） in pulmonary alveolar macrophage（ PAM） cells of rats. Methods DPSCs were cultured in the complete culture medium,and their supernatants at passage 6 were collected after serum-free medium treatment for 24 hours. Exosomes were extracted and purified with ultracentrifugation. Rat PAM NR8383 was cultured in 12-well plate and treated with LPS of 1 μg/ml alone or together with exosomes. The supernatants were then collected at 0,6,12 and 24 h respectively after treatment. Inflammatory cytokine levels of tumor necrosis factor-α（ TNF-α） and interleukins（ IL-1β and IL-6） in the supernatant were measured by ELISA assay and the expression and phosphorylation level of MAPK（ p44/42）,NF-κB and IκBα in cell lysates were detected with Western-blotting. Results Compared with control group,the content of TNF-α,IL-1β and IL-6 increased significantly in LPS group（ P〈0. 05）,which indicated that the inflammatory cell model was induced successfully. The levels of TNF-αand IL-1β were obviously attenuated after a high doses of exosomes treatment（ P〈0. 05）,and the expression of IL-6 was markedly suppressed after low and high doses of exosomes treatment（ P〈0. 05）,compared with the group of LPS treatment alone. The phosphorylation of NF-κB,IκBα and p44/42 was significantly inhibited after treatment with the DPSCs-derived exosomes. Conclusion DPSCs-derived exosomes may have a potential protective effect on LPS-induced ALI,and the underlying mechanism is that the activity of MAPK（ p44/42） and NF-κB/IκBα pathways are eliminated by DPSCs-derived exosomes.
作者
苏晓磊
汪坤
刘月
肖凤君
吴祖泽
张庆林
靳继德
SU Xiao-lei;WANG Kun;LIU Yue;XIAO Feng-jun;WU Zu-ze;ZHANG Qing-lin;JIN Ji-de(Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China)
出处
《军事医学》
CAS
CSCD
北大核心
2018年第2期130-137,共8页
Military Medical Sciences
