EPC来源的外泌体介导肺微血管内皮细胞重编程对BPD的防治作用及机制研究

To promote the development of pulmonary vessels to alveolarization is the the forefront of research in prevention of bronchopulmonary dysplasia (BPD). Endothelial progenitor cells (EPCs) are "seed" cells in promoting the development of pulmonary vascular. Our previous studies have shown that simply increasing the number of EPC targeting prevention of BPD had poor results, however, EPC-derived angiocrine factors could mediate the proliferation of lung epithelial cells. But there are too much angiocrine factors. Exosome derived from EPC (EPC-Exosome) is an important member in family of angiocrine factors, new research suggests EPC-Exosome includes miRNAs, which can be transmitted to the receptor cells like endothelial cells, reprogramming endothelial cells. This project intends to study the EPC-Exosome biological characteristics, detect miR-126 and miR-296 by gene sequencing and qRT-PCR; detect CXCR4, CXCR7, VEGFR2，Akt and eNOS expression in pulmonary microvascular endothelial cells (PMECs) and functional changes of PMECs after communication between EPC-Exosome and PMECs; and effects on alveolar epithelial cells II by reprogrammed PMECs with EPC-Exosome. This project also study the effcts on the number of lung microvascular and alveolars in neonatal hyperoxia exposed rats by injecting EPC-Exosome. This approach may also optimize cell-based treatment approaches and contribute to the development of new therapeutic strategies to prevent BPD.

促进肺血管发育介导肺泡化是防治支气管肺发育不良（BPD）的研究前沿，内皮祖细胞（EPC）是促进肺血管发育的“种子”细胞。本课题组前期研究表明单纯以提高EPC 数目为目标的EPC移植防治BPD 效果欠佳，而EPC旁分泌信号能介导肺上皮细胞增殖。EPC 来源的外泌体（EPC-Exosome）是旁分泌信号的重要成员，最新研究表明EPC-Exosome能介导内皮细胞重编程，促进血管生成和器官再生。本研究拟开展如下研究：（1）研究大鼠来源EPC-Exosome的生物学特性和EPC-Exosome介导的肺微血管内皮细胞（PMECs）重编程作用；（2）研究重编程后的PMECs与大鼠肺泡II型上皮细胞的交互作用；（3）通过动物实验，研究外源性输入EPC-Exosome对新生大鼠高氧暴露BPD模型肺微血管数目和肺泡化的影响。为进一步优化EPC 细胞移植治疗方法，发展BPD 防治新策略提供全新思路和实践依据。

促进肺血管发育介导肺泡化是防治支气管肺发育不良（BPD）的研究前沿，内皮祖细胞（EPC）是促进肺血管发育的“种子”细胞，EPC-Exosome能介导内皮细胞重编程，促进血管生成和器官再生。本研究以大鼠来源EPC-Exosome为研究对象，研究的生物学特性和EPC-Exosome的生物学特性及其介导的肺微血管内皮细胞（PMECs）重编程作用。通过建立BPD动物实验，研究外源性输入EPC-Exosome对新生大鼠高氧暴露BPD模型肺微血管数目和肺泡化的影响。主要实验结果：（1）EPCs分离培养和鉴定：所得细胞培养至第5天形成小集落样，培养至第7天左右出现典型的梭形、铺路石样改变；所得细胞具有摄取Dil-ac-LDL和结合FITC-UEA-1的功能;可在Matrigel上形成管腔样结构；表达CD34、VEGFR2表面抗原。（2）EPCs-EXO提取和鉴定：透射电子显微镜下外泌体均呈双凹杯托状。蛋白免疫印迹法可检测到EPCs-EXO表达标记蛋白CD63和TSG101。试剂盒提取的外泌体平均粒径99.7±31.2nm，粒子浓度为6.85×108±8.5×107个/mL；超高速离心法提取的外泌体平均粒径181.3±61.2nm，粒子浓度为2.87×108±2.97×107个/mL。（3）EPCs-EXO含调控血管新生的miR-126 和miR-296。（4）PMVECs可摄取培养环境中的EPCs-EXO。（5）EPCs-EXO可促进高氧暴露PMVECs的细胞增殖、细胞迁移和细胞成管能力。（6）EPCs-EXO可改善高氧暴露PMVECs表达VEGF、VEGFR2、eNOS、 MMP14。（7）大鼠腹腔注射硫酸博来霉素可产生类似BPD病理改变，使肺泡简化和肺内血管密度降低。