鞠志海，王 晨，马金辉，于 洁，乔叶薷，黑飞龙



iPSC外泌体载体肺血管内皮细胞及肺泡Ⅱ型上皮细胞摄入研究

鞠志海，王 晨，马金辉，于 洁，乔叶薷，黑飞龙

［摘要］：目的 全身炎症反应在体外循环致肺损伤过程中起关键作用。外泌体载体能否被肺脏靶细胞有效摄入是急性肺损伤(ALI)基因治疗的关键。方法 超速离心法提取体外培养的iPS细胞外泌体,Western Blot鉴定其特征蛋白表达。PKH26标记的外泌体与肺微血管内皮细胞和肺泡Ⅱ型上皮细胞体外共培养,免疫荧光共聚焦技术检测靶细胞对外泌体的吸收、摄取情况。结果 iPS细胞来源的外泌体可以被肺微血管内皮细胞和肺泡Ⅱ型上皮细胞有效摄入。结论 iPS细胞外泌体可以被肺脏靶细胞有效摄入,是ALI基因治疗的理想载体。

［关键词］：急性肺损伤；iPS细胞；外泌体；载体；摄入

Study on uptake of exosomes derived from iPSC in pulmonary microvascular endothelial cells and typeⅡalveolar epithelial cells

Ju Zhi-hai,Wang Chen,Ma Jin-hui,Yu Jie,Qiao Ye-ru,Hei Fei-long
Department of Cardiopulmonary Bypass,Fu Wai Hospital,CAMS and PUMC,National Center for Cardiovascular Diseases,Beijing 100037,China
Corresponding author:Hei Fei-long,Email:heifeilong@ 126.com

［Abstract］：Objective Systemic inflammatory response plays a vital role in lung injury caused by CPB.The success of gene therapy toward to ALI is mainly depended on whether an exosome vehicles could be absorbed by the target cells in lung.Methods Exosomes were isolated from iPS cells(iPS-Exo) derived from renal tubular epithelial cells using ultracentrifugation and characterized by western blotting and then labeled with PKH26 and cocultured with pulmonary microvascular endothelial cells(PMVECs) or typeⅡalveolar epithelial cells(A549),Using laser scanning confocal microscopy to decide the uptake of these exosomes.Results iPS-Exo could be absorbed by both PMVECs and A549 in vitro.Conclusion The iPS-Exo may be considered as an ideal carrier since they could be delivered to the target cells in lung successfully.

［Key words］： Acute lung injury；iPS cell；Exosomes；Carrier；Uptake

体外循环(cardiopulmonary bypass,CPB)手术期间,肺是受损最为常见和损伤严重的器官之一,重者可表现为急性肺损伤(acute lung injury,ALI)[1]。肺损伤机制目前尚未明了,学者研究认为全身炎症反应在CPB肺损伤过程中发挥重要作用[2],特别是NLRP3(nucleotide-binding-domain,leucine-rich repeat domain containing protein 3,NLRP3)炎性小体对ALI炎性调控作用十分关键[3-4]。siRNA( small interfering RNA)沉默NLRP3炎性小体可有效控制炎症反应,减轻组织损伤[5]。iPS细胞外泌体(exosomes)是非常理想的siRNA载体,其能否被肺脏的靶细胞高效吸收和摄取成为ALI精准基因治疗的关键。

1 试剂与方法

1.1 主要试剂 iPS细胞由本课题组前期研究获得[6],肺泡Ⅱ型上皮细胞(A549购自ATCC),PSCeasy多潜能干细胞培养基(赛贝生物),肺微血管内皮细胞及ECM培养基(Sciencell),PKH26(Mini 26,Sigma),Phalloidin-FITC(P5282,Sigma),FITCWGA(ab20528,Abcam),抗CD63抗体(ab59479,Abcam),抗TSG101抗体(ab83,Abcam)。

1.2 材料与设备 超速离心管(Beckman)、Optima L-100XP超速离心机(Beckman)、Leica SP8激光扫描共聚焦显微镜(Germany)。……