miRNA-150过表达MSCs促进TNTs线粒体传输抗肺泡巨噬细胞凋亡改善LPS诱导ARDS的作用研究

Our previous studies revealed that alveolar macrophage (AM) apoptosis in early stage plays a crucial role in acute respiratory distress syndrome (ARDS) pathogenesis. And mitochondrial donation through newly discovered structure-Tunneling Nanotubes (TNTs) is main therapeutic mechanism of mesenchymal stem cells (MSCs) for AM apoptosis as well as ARDS. However multiple clinical studies have suggested that further research is still be needed to find new strategies to enhance efficacy of MSCs therapy. Our preliminary data showed that the expression of miRNA-150 was up-regulated accompanied with the activation of PI3K-Akt during the formation of TNTs in MSCs. Here we hypothesize that miRNA-150 over expression could activate of PI3K-Akt and subsequently cause the formation of TNTs, which lead to reduce AM apoptosis and finally ameliorate ARDS. In this study, first, miRNA-150 & PI3K genetic overexpress and silence technique will be employed to elucidate the possible mechanism of miRNA-150 regulates the formation of TNTs. Then based on the results of first part, we will develop an innovative strategy (MSCs with miRNA-150 genetic overexpressed) to enhance the mitochondrial donation of MSCs. At last we will examine the protective effect against apoptosis in AM and ARDS in mice after LPS stimulation. These results will verify whether miRNA-150 genetic overexpressed will make MSCs be more efficacy. This study is expected to provide theoretical basis for MSCs therapy using in clinic practice.

申请人发现肺泡巨噬细胞（AM）早期凋亡是LPS诱导急性呼吸窘迫综合征（ARDS）的重要发病机制。研究发现通过新近发现的隧道纳米管（TNTs）传输线粒体进而减少AM早期凋亡是间充质干细胞（MSCs）的主要作用，但临床研究发现未经基因修饰的MSCs治疗效率较低。预实验提示MSCs 的TNTs形成伴随miRNA-150的上调和PI3K-Akt的活化。申请人推测MSCs内miRNA-150表达上调，通过活化PI3K-Akt，促进TNTs形成，进而减少LPS诱导AM早期凋亡，最终缓解ARDS。本课题组通过抑制或过表达MSCs内miRNA-150和PI3K的表达，研究miRNA-150对TNTs形成的调控作用。构建miRNA-150过表达的MSCs，通过细胞和动物实验验证miRNA-150过表达的MSCs有更强的抗AM凋亡作用，对ARDS有更高的治疗效率。本课题将为MSCs进入临床广泛使用奠定基础。

通过基因修饰骨髓充质干细胞(BMSCs)课增强BMSCs对内毒素（LPS）诱导的肺泡巨噬细胞（AMs）早期凋亡的抑制作用，对临床ARDS治疗有重大临床价值。.本课题研究基因修饰的BMSCs线粒体传递能力对LPS诱导小鼠ARDS和肺泡巨噬细胞线粒体功能和凋亡的治疗效果，重点探讨micro-150表达对BMSCs线粒体传输能力的影响，并阐述其分子调控的机制。.本研究从动物和细胞水平验证，BMSCs改善LPS诱导小鼠肺内炎症，水肿及肺泡上皮细胞氧化应激，并进一步在细胞水平中发现，BMSCs传递线粒体改善LPS诱导巨噬细胞线粒体功能和氧化应激减少巨噬细胞凋亡是减轻肺部炎症反应的重要途径，BMSCs中miR-150下调通过增加线粒体传递，增强BMSC对LPS刺激肺泡巨噬细胞线粒体功能和凋亡的治疗作用，直接靶向BMSCs中MTCH2基因是其调控途径，LPS刺激BMSCs中miR-150下调，MTCH2基因上调是BMSCs增加线粒体传递，增加对LPS刺激肺泡巨噬细胞线粒体功能和凋亡疗效的重要机制。.本课题为以线粒体导向的BMSCs基因修饰提供了新的理论依据，同时，微小RNA是一种成熟经济的基因修饰手段，通过BMSCs基因修饰可改善微小RNA不稳定无法用于临床治疗的技术难点，为BMSCs治疗提供了有效途径。