iPSc外泌体介导siRNA靶向沉默NLRP3炎性体治疗急性肺损伤的作用及机制研究

There is no effective method for the treatment of ALI until now. Inflammation plays a vital role in the pathophysiological process of ALI. Research shows that using siRNA to silence NLRP3 inflammasome could alleviate inflammatory cascade reaction. An ideal carrier is key of the genetic therapy for ALI. The exosomes derived from iPSc show a prominent advantage as a carrier for siRNA delivery. We have already extracted iPSc exosomes successfully and the further study show that the iPSc exosomes could be uptake in the pulmonary microvascular endothelial cells and alveolar type II epithelial cells. Therefore, a hypothesis is proposed that using iPSc exosomes loading specific siRNA to silence NLRP3 inflammatory body may alleviate inflammatory cascade reaction and treat of ALI. To verify our hypothesis, we plan to culture iPSc in vitro, extract exosomes and load specific siRNA and then study the efficiency of this technology on the treatment of whole and cell model for ALI.Then investigate the mechanism under the inhibition of signaling pathways related to NLRP3 inflammatory body using the culture of alveolar macrophages in vitro and estimate the efficiency of the RNA interference gene therapy on ALI at a cellular level. Elucidating the iPSc exosomes mediated RNA interference on the suppression of NLRP3 inflammatory body from the upstream of the signal pathway and reducing inflammation during the process of ALI could provide a new method and theory for the prevention and control of ALI.

ALI防治仍缺乏有效方法，炎性反应是其重要的发病机制。新近研究显示siRNA沉默NLRP3炎性体可减轻炎性级联反应，其给药载体选择是其发挥ALI基因治疗的关键。iPSc外泌体作为siRNA载体，优点突出。课题组在提取iPSc外泌体的基础上，进一步研究发现其可被肺微血管内皮细胞和肺泡二型上皮细胞有效摄入。据此假设，iPSc外泌体可通过装载特异性siRNA被肺脏靶细胞摄取，进而沉默NLRP3炎性体，减轻炎症级联反应，抑制ALI。为验证假设，拟培养iPSc，提取外泌体装载特异性siRNA；然后在小鼠ALI模型上从整体水平验证其抑制炎性反应，减轻ALI的有效性；再以肺泡巨噬细胞为靶点在细胞水平探讨RNAi基因治疗通过NLRP3信号通路抑制炎性反应的机理。从而论证iPSc外泌体介导的RNAi技术从上游环节抑制NLRP3炎性体激活、减轻ALI炎症反应的机理和效果，为ALI的防治提供新方法及理论依据。

急性肺损伤(Acute lung injury，ALI)是一种以肺血管内皮细胞和肺泡上皮细胞广泛受损伤为特征的失控性炎症反应。临床出现严重的低氧血症和(或)高碳酸血症，严重者可致急性呼吸窘迫综合征(Acute respiratory distresssyndrome，ARDS)，病死率高达40％，而肺泡巨噬细胞在肺损伤的发生过程中起着重要作用。而目前干细胞治疗给肺损伤的治疗带来新曙光。越来越多的研究表明干细胞以外泌体的旁分泌作用方式起治疗作用。胞外囊泡是细胞旁分泌作用的一种重要方式，其包括外泌体（exosomes）、微囊泡（microvesicles）及凋亡小体。但其在肺损伤中的保护及致病机制仍未明确。因而本项课题采用诱导多潜能干细胞来源的外泌体转载siRNA观察其对肺损伤的治疗作用以及探究肺泡巨噬细胞来源的微囊泡在肺损伤中的作用机制。本项研究成功提取尿路上皮细胞分化而来的诱导多潜能干细胞（Induced pluripotent stem cells, iPSCs），通过差速梯度离心获取iPSCs来源的外泌体，并将外泌体通过电转方式装载小干扰RNA（siRNA）后作用于肺微血管上皮细胞，结果表明肺微血管上皮细胞摄取iPSC来源转载siRNA的外泌体，而siRNA-外泌体抑制了肺微血管上皮细胞ICAM-1的转录及蛋白表达。此外，通过大量培养炎性肺泡巨噬细胞并在刺激早期获取其微囊泡，酶联免疫吸附实验表明此微囊泡主要表达TNF-α炎性介质。肺泡巨噬细胞来源的炎性微囊泡明显增加了肺损伤程度并导致肺上皮细胞损伤。同时，炎性微囊泡导致肺水肿液清除率降低，增加了肺组织湿/干重比例及肺泡灌洗液中炎性细胞浸润，并降低了肺泡上皮细胞钠通道蛋白表达。诱导多潜能干细胞来源外泌体的治疗作用以及肺泡巨噬细胞来源微囊泡的损伤作用均表明胞外囊泡包括外泌体及微囊泡在肺损伤中起着重要作用，这为肺损伤的防治提供了更多的方向。