Gpr153通过cAMP/PKA通路调控特络细胞旁分泌在急性肺损伤修复中的作用和机制

Acute lung injury is a common pulmonary severe disease with high mortality, and the current therapies are lack of efficacy. Our prior studies found that telocytes exsit in pulmonary interstitium and be able to modulate inflammation and help lung injury repair through some paracrine cytokines. In addition, our study from genomic analysis reveals that telocytes highly express Gpr153 (G protein-coupled receptor 153) gene. Thus, we proposed this hypothesis: Gpr153 gene could enhance the paracrine effects of telocytes through cAMP/PKA pathway, thus help modulate inflammation repair the injured lung. In order to validate this hyposis, we will discuss the role and mechanism of Gpr153 gene on the paracrine effects of telocytes through cAMP/PKA pathway in acute lung injury by means of ELISA, real time-PCR, Western blot, lentivirus vector transfection and RNA interference in mice primary telocytes, mouse alveolar epithelial cells and LPS injured mice models. By focusing on a new viewpoint of modulating telocytes’ paracrine effects by Gpr153 gene, this project will further reveal the mechanisms of the onset and repair in lung injury, and try to provide experimental and theoretical foundation for the further in vivo study in the treatment of lung injury with telocytes.

急性肺损伤是常见呼吸道重症疾病，死亡率很高，现有的治疗方法疗效有限。我们前期的研究发现，特络细胞位于肺组织间质中，可能参与肺泡上皮细胞的损伤、修复和再生。对特络细胞的基因谱分析发现其高表达Gpr153（G蛋白偶联受体153）基因。为此，我们提出假说：Gpr153基因可能通过cAMP/PKA信号通路调控特络细胞旁分泌、炎症反应和肺损伤修复。为了验证这一假说，我们将通过小鼠原代特络细胞、小鼠肺泡Ⅱ型上皮细胞和LPS肺损伤模型，采用real time-PCR、Western blot和ShRNA干扰等手段，从分子、细胞和动物水平等多方面探讨Gpr153基因调控特络细胞旁分泌在肺损伤修复中的作用，明确其通过cAMP/PKA通路调控旁分泌的机制。本研究将从Gpr153调控特络细胞旁分泌这个新视点着手，为揭示急性肺损伤的发生和修复机制奠定基础，也为深入开展以特络细胞为基础治疗肺损伤奠定实验和理论基础。

急性肺损伤是常见呼吸道重症疾病，死亡率很高，现有的治疗方法疗效有限。我们前期的研究发现，特络细胞（TCs）可能参与肺泡上皮细胞的损伤、修复和再生，且基因谱分析显示其高表达Gpr153（G蛋白偶联受体153）基因。我们提取小鼠原代肺TCs，建立小鼠肺TCs系，通过慢病毒转染和shRNA 干扰构建Gpr153 基因高表达或低表达的TCs，进一步利用体内体外急性肺损伤模型，从分子、细胞和动物水平等多方面探讨Gpr153基因调控特TCs旁分泌在肺损伤修复中的作用。TCs促进了LPS处理的小鼠肺泡上皮细胞系MLE-12的增殖，减轻了小鼠急性肺损伤模型肺部炎症，Gpr153高表达的TCs进一步减轻了肺部炎症因子的表达，而敲除该基因的TCs不再具有该作用。体外实验发现LPS刺激的TCs分泌IL6和IL8水平显著降低，Gpr153敲除的TCs其IL1和TGFβ1表达增加，并抑制了MLE-12细胞增殖，而SQ22536（腺苷酸环化酶抑制剂）作用后逆转了敲除Gpr153的TCs的作用。因此，Gpr153通过腺苷酸环化酶通路调控TCs旁分泌从而维持其急性肺损伤修复的作用，并明确了TGFβ1是LPS刺激后TCs产生的重要炎症调控因子，证明了对TCs的细胞增殖和周期有重要的调控作用[Dongli Song, et al. 2019;17(1):431]。本研究所构建的小鼠肺TCs细胞系为TCs的研究拓展了更大的空间，研究结果为揭示急性肺损伤的发生和修复机制奠定基础，为深入开展以TCs为基础治疗肺损伤奠定实验和理论基础。