CXCL10促进肺泡干细胞增殖作用机制及其在急性肺损伤中的保护作用研究

Acute lung injury (ALI) is critical respiratory disease associated with high mortality. The management for ALI still relies on general supportive measures without any effective medicine and intervention so far. The principle pathophysiological change in ALI is the diffuse alveolar damage caused by various injuries to the cells. The natural history of ALI consists of acute inflammation, subacute proliferative and fibrotic phases. Most previous studies focused on counteracting the inflammation, but most related drug clinical trials failed to demonstrate any clinical benefit. The alveolar epithelial cell type 2 (AEC2) is the stem cell of alveolar epithelia and plays an important role in the epithelial regeneration of ALI. How to increase the regenerative ability of AEC2 is the key target of future ALI study. CXCL10 is expressed in injured tissues, where it plays critical roles in modulating multiple cellular functions. We previously found that CXCL10 could promote AEC2 proliferation, independent of CXCR3. We plan to sort AEC2 by FACS followed by clonal 3D organoid culture. The colony formation efficiency (CFE) between WT AEC2 and SPC-CXCL10 AEC2 will be compared. We will construct the CXCL10 mutants deficiency of CXCR3 binding ability, and compare the CFE of WT AEC2 with exogenous CXCL10 recombinant protein, CXCL10 monoclonal antibody, or the CXCL10 mutants. To identify the molecular markers of the proliferative effect of CXCL10 on AEC2, we will screen for the significant changed genes in AEC2 stimulated by CXCL10 employing the mRNA sequence method. We will employ SPC-CXCL10 transgenic and WT mice to establish the influenza virus induced infectious and bleomycin induced non-infectious ALI mouse model, and compare the survival rate and fibrosis, measure lung function by BUXCO, analyze the lung inflammation of immune cell infiltration and BALF cytokines production, analyze the population of AEC2 using the flow cytometry analysis and proliferation of AEC2 using immunofluoresence staining of Ki67, to confirm the regenerative ability and protective effects of endogenous CXCL10 in vivo. We will further test the protective effects of intramuscular injection of the CXCL10 mutant and transplantation of WT or SPC-CXCL10 AEC2, on the two ALI mouse models, by comparing the survival rate, lung function, inflammation, fibrosis, and AEC2 proliferation. We hypothesized that besides playing a role in lung inflammation, CXCL10 also is critical in regulating lung AEC2s repair during influenza virus or bleomycin induced lung injury. The injuries to the lung induce AEC2s to express CXCL10, which acts on AEC2s in an autocrine manner, promoting the regeneration and proliferation of AEC2s after injury in ALI. Understanding the regenerative capacity of CXCL10 on AEC2s is of considerable practical and therapeutic interest and the accomplishment of the current study will provide new therapeutic targets and potential treatment to protect ALI.

急性肺损伤(ALI)是病死率极高的危重症，无特异治疗手段。肺泡干细胞增殖决定ALI转归，如何提高其增殖活性是治疗关键。我们前期发现CXCL10对肺泡干细胞的增殖具重要作用，且不依赖CXCR3。在此基础上，本项目拟在流感病毒和博莱霉素模型下，揭示内源或外源性CXCL10及CXCR3结合缺陷型突变体，对肺泡干细胞在体内或体外的增生作用；二代测序筛查与增殖相关的基因，并检测增殖相关信号通路的变化，明确CXCL10对肺泡干细胞的增殖机制；建立流感病毒、博莱霉素导致的感染性、非感染性ALI小鼠模型，比较①WT与SPC-CXCL10转基因小鼠之间②注射突变体③移植WT或转基因小鼠的肺泡干细胞，两种ALI模型死亡率、肺功能、炎症、纤维化、肺泡干细胞数量和增生的改变，验证内源性CXCL10、突变体、肺泡干细胞移植对两种ALI模型的保护作用。该成果将为ALI提供新治疗靶点和可能保护药物，改善ALI治疗现状。

急性肺损伤(ALI)是病死率极高的危重症，目前临床仍无特异治疗手段。肺泡II型上皮细胞（AEC2）是维持和修复肺泡上皮的干细胞，其增殖是ALI转归的关键，而提高AEC2的增殖活性是治疗ALI的重要目标。趋化因子CXCL10在损伤组织中表达并调节多种细胞的功能。然而，CXCL10在AEC2再生过程中的作用尚知之甚少。本项目在前期研究基础上，提出肺损伤诱导AEC2表达CXCL10并促进其再生和增殖，从而保护ALI的假设。我们在多种小鼠ALI模型中，均发现CXCL10可显著升高。应用在AEC2特异性过表达CXCL10的SPC-CXCL10的转基因小鼠，对小鼠肺单细胞匀浆进行流式细胞术分析显示，与WT小鼠相比，SPC-CXCL10转基因小鼠在损伤修复期肺内AEC2细胞群显著增加。通过3D培养实验，发现内源性SPC-CXCL10转基因小鼠过表达的CXCL10能够使AEC2的克隆形成能力显著增强。外源性CXCL10重组蛋白质能促进AEC2增殖，而CXCL10 mAb抑制AEC2增殖。.应用3D培养实验，我们发现CXCR3缺陷小鼠的CXCL10再生功能与CXCR3无关，而受体酪氨酸激酶TrkA则与CXCL10的再生效应密切相关。Western印迹显示TrkA在暴露于CXCL10时被磷酸化激活，其下游MAPK信号通路也被激活。对CXCL10处理的原代人肺SAEpi细胞进行RNA-seq并分析、富集差异表达基因，发现CXCL10刺激下表达受影响的基因，主要集中在细胞生长与脂代谢等通路。细胞生长通路可能由TrkA介导的MAPK信号通路激活所引发。.为探讨CXCL10在肺损伤和修复中的作用，将博莱霉素气管内注射SPC-CXCL10小鼠和WT小鼠。与WT小鼠相比，SPC-CXCL10转基因小鼠的存活率明显升高，但是，胶原染色与羟脯氨酸的含量没有显著差异。小鼠存活率的升高，可能是因为博莱霉素导致肺细胞损伤之后，与WT小鼠相比，SPC-CXCL10转基因小鼠的AEC2具有更强的恢复期再生能力，增殖并分化产生更多的AEC1修复肺气血屏障，从而恢复肺的换气功能，提高小鼠存活率。.因此，我们认为CXCL10在调节肺损伤后肺AEC2修复中起重要作用。这提示CXCL10促进AEC2增殖的特点，可以作为治疗ALI的潜在靶点和可能保护药物，改善ALI治疗现状。