间充质干细胞外泌体对肺脏上皮钠离子转运的调控作用研究

The Research about the Regulation of Mesenchymal Stem Cell Exosomes on Sodium Transport of Lung Epithelium

该文讨论了小鼠骨髓间充质干细胞来源的外泌体(bone mesenchymal stem cell-exosome,BMSC-exo)对肺损伤引起的肺泡上皮钠离子转运障碍的调控。从BMSCs的条件培养基中分离外泌体,利用透射电镜技术对其形态结构以及大小进行了鉴定;对培养的经典肺上皮细胞系H441细胞分别给予脂多糖或外泌体处理,应用qRT-PCR和Western blot技术检测了H441细胞中钠离子通道在mRNA和蛋白水平的表达情况。此外,研究结果表明,LPS处理的H441细胞中miR-199a-3p的表达明显降低;与单独应用LPS组相比,浓度为20μg/mL的外泌体处理组中miR-199a-3p的表达显著性升高;和阴性对照组(NC)相比,转染miR-199a-3p mimic的H441细胞中α-、γ-ENaC的表达明显升高,而和inhibitor NC组相比,miR-199a-3p inhibitor组的α-、γ-ENaC的表达则明显降低。网站预测结果显示,哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是miR-199a-3p的靶蛋白,miR-199a-3p mimic组的mTOR蛋白的表达明显低于NC组;miR-199a-3p inhibitor组和inhibitor NC组相比,mTOR的表达显著性升高。以上结果表明,BMSC-exo可能经miR-199a-3p参与mTOR通路调节肺泡上皮细胞中钠离子通道的表达来促进肺脏上皮离子转运,进而可能促进病理条件下的肺脏液体清除,参与临床急性肺损伤等相关水肿性肺疾病的治疗。

This study discussed the regulation of BMSC-exo(bone mesenchymal stem cell-exosome)derived from mouse on alveolar epithelial sodium transport disorders caused by lung injury.The exosomes from the conditioned medium of BMSCs were isolated and their morphology,structure and size were identified by transmission electron microscopy.The cultured H441 cells,the classical lung epithelial cell line,were treated with lipopolysaccharide or exosomes,respectively.The expression of sodium ion channels at mRNA and protein levels in H441 cells separately was detected by qRT-PCR and Western blot.The results of this study showed that the protein expression levels ofα-,γ-ENaC in the group combined exosomes and LPS were significantly increased compared with the LPS-treated group.Besides,the mRNA expression levels of theα-,β-,andγ-subunits were also significantly increased.In addition,our results also showed that the expression of miR-199a-3p in LPS-treated H441 cells was significantly decreased.Compared with the LPS group,miR-199a-3p expression was significantly increased in the exosome treatment group at 20μg/mL.Compared with negative control group,the expression levels ofα-andγ-ENac in H441 cells transfected with miR-199a-3p mimic were significantly increased,while those of miR-199a-3p inhibitor group were significantly decreased compared with inhibitor negative control group.The prediction of the website showed that mTOR(mammalian target of rapamycin)was the direct target of miR-199a-3p.mTOR protein expression of the group transfected with miR-199a-3p mimic was significantly lower than the negative control group,while mTOR expression of the group transfected with miR-199a-3p inhibitor was significantly increased compared with the inhibitor negative control group.The above results suggest that BMSC-exo may participate in the mTOR pathway through miR-199a-3p to regulate the expression of sodium ion channels in alveolar epithelial cells to promote the ion transport in pulmonary epithelium,which may further improve the clearance of pulmonary fluid under pathological conditions and participate in the treatment of clinical acute lung injury and other related edema lung diseases.