肺源性微囊泡诱导移植间充质干细胞定向分化治疗肺纤维化

The death of alveolar epithelial cells (AECs) and the destruction of the cell junctions are considered as initial factors of the pulmonary fibrosis (PF). Thus, the effectively repair of the destroyed and the dead AECs which result in the rebuilt of the cell junction integrity at the early stage should be a feasible way to prevent the PF process. Our previous investigations have proved that mesenchymal stem cells (MSCs) can differentiate into AECs. And we can suppress the proceeding of the PF by MSCs engraftment and inducing them differentiating into AECs which may fill the niches of the dead AECs. However, the proportion of the MSCs differentiated into AECs was extremely low. A dramatic enhancement of the AECs differentiation proportion of MSCs exists as a bottleneck problem. Microvesicles (MVs), which contain nucleic acids and proteins , are an important way of cell-to-cell communication. Injured cells can send messages to stem cells through MVs delivery and induce the recipient stem cells differentiating into the same phenotype and migrating to the injured tissues to repair later on. Our in vitro study will aim on the differentiation proportion enhancement of MSCs by co-culture with the lung derived microvesicles (LDMVs). And in vivo study will try to significantly alleviate PF of the SD rat model by MSCs lung engraftment which are pretreated with LDMVs in hope that more MSCs can differentiate into AECs to exert their healing effects. The results of our research may not only be important in revealing the mechanisms of PF but also provide substantial experimental references of PF treatment through regeneration properties of stem cells .

肺泡上皮细胞(AEC)死亡、细胞间连接结构破坏是肺纤维化(PF)发生的始动环节。早期高效地修复受损及死亡的AEC，重建其连接的完整性是治疗PF的有效途径。我们的研究证明：通过肺内移植MSC进而分化为AEC以填补死亡细胞龛位能有效地抑制PF的进展。然而，移植MSC向AEC分化的效率极低。此为有效利用MSC进行肺组织再生修复的瓶颈问题。微囊泡(MV)内含核酸、蛋白等信息物质，是细胞间信息传递的重要途径。受损细胞通过释放MV将自身信息传递给干细胞，诱导其向受损细胞类型分化以参与组织重建。本研究将通过肺源性MV(LDMV)与MSC共培养，体外证实LDMV对MSC向AEC分化的促进作用；并将经LDMV预处理的MSC用于PF模型大鼠的肺内移植治疗，使移植的MSC在肺内更多地向AEC分化，更好地参与受损肺组织的早期修复，提高治疗效率。本研究将为利用干细胞组织再生特性治疗PF提供实验依据及理论支持。

本研究旨在利用肺源性微囊泡对大鼠来源的骨髓间充质干细胞向肺泡上皮细胞进行诱导分化，并期望通过外源性肺泡细胞囊泡的诱导作用，使得骨髓间充质干细胞获得肺泡细胞的功能，并参与到肺组织损伤后肺泡细胞的修复进程中，一方面能够减轻肺组织损伤，同时能够更好的对受损伤的肺组织进行及时有效的外源性干细胞修复，减轻后期肺组织纤维化的发生、发展。为验证上述研究构想，我们通过提取大鼠肺泡上皮细胞外泌体对大鼠骨髓间充质干细胞进行诱导转分化尝试。研究结果表明：肺泡上皮细胞分泌的外泌体无法在体外将骨髓间充质干细胞转分化为肺泡上皮样细胞。后研究组将实验方案调整为利用骨髓间充质干细胞外泌体对博来霉素损伤下大鼠肺泡巨噬细胞的激活及计划状态进行调节，并以此为基础，减轻肺损伤后纤维化的发生及发展。研究结果表明：IL-1β、TNF-α及TGF-β1在加入博来霉素干预后的早期时相，其表达水平明显增高，而 12小时后，其表达水平恢复正常；加入骨髓间充质干细胞培养上清提取的外泌体，虽IL-1β、TNF-α水平有所降低，但差异无统计学意义。与此同时，将传统的二维细胞培养改为利用FiberCell Culture system的三维培养方式进行细胞培养。通过该系统的使用，我们建立了一种高效、大量获取外泌体的实验方法，并对所获得的外泌体进行了定性及定量检测。研究结果表明：该方法可大量扩增间充质干细胞，并维持间充质干细胞在较高的数量水平（1*10^9 MSCs）稳定生长。并可通过系统持续收集间充质干细胞外泌体。目前我们正将该提取自该系统的外泌体用于肺泡巨噬细胞博来霉素诱导刺激实验。