CD3+CD28-T细胞通过外泌体miR-483-5p促进人肺成纤维细胞增殖及转分化参与IPF发病的机制研究

Idiopathic pulmonary fibrosis (IPF) is a rare chronic disease associated with aging. Its prognosis is extremely poor and pathogenesis is unclear. Immunosenescence characterized by an increase in the proportion of CD3+CD28-T cells promotes fibroblast proliferation and participates in tissue fibrosis, but its role in pulmonary fibrosis needs further exploration..Our study found that the proportion of CD3+CD28-T cells in peripheral blood of patients with IPF was significantly increased. CD3+CD28-T cells were cultured from the peripheral blood of patients with IPF. Cell culture and exosomes increased the proliferation of human lung fibroblasts (HLF) and increased the expression of α-SMA and Collagen I. High-throughput sequencing of miRNAs in CD3+CD28-T exosomes from IPF patients showed a significant increase in miR-483-5p compared to the normal control group. Therefore, we propose that CD3+CD28-T cells can act on HLF cells by exosome carrying miR-483-5p, promote proliferation and transdifferentiation of HLF cells, and participate in pulmonary fibrosis. This subject will study the molecular mechanism of CD3+CD28-T cells regulating fibroblast proliferation and transdifferentiation in the process of pulmonary fibrosis through human, cell and animal experiments.

特发性肺纤维化（IPF）是一种罕见的与衰老相关的慢性肺病，预后极差，发病机制不清楚。以CD3+CD28-T细胞比例升高为特征的免疫衰老可促进成纤维细胞增殖，参与组织纤维化过程，但其在肺纤维化中的作用需进一步探索。.我们研究发现IPF患者外周血CD3+CD28-T细胞比例明显升高。从IPF患者外周血中提取CD3+CD28-T细胞培养，细胞培液及细胞外泌体可使人肺成纤维细胞（HLF）增殖增加，α-SMA及Collagen I的表达增加。对IPF患者CD3+CD28-T细胞外泌体行miRNA高通量测序显示miR-483-5p较正常对照组显著升高。因此我们提出CD3+CD28-T细胞通过携带miR-483-5p的外泌体靶向作用于HLF细胞，促进HLF细胞增殖及转分化，参与肺纤维化过程。本课题将从人体、细胞、动物三方面研究CD3+CD28-T细胞在肺纤维化进程中调控成纤细胞增殖及转分化的分子机制。

特发性肺纤维化是一种衰老相关疾病，是一种以肺泡上皮细胞损伤、成纤维细胞大量增生聚集及细胞外基质沉积为主要病理特点的罕见的慢性肺疾病，诊断后3-5年中位生存率仅为50%。免疫衰老可参与调节多种衰老相关疾病，衰老T淋巴细胞促进成纤维细胞增殖，参与组织纤维化过程。特发性肺纤维化患者中免疫衰老（CD3+CD28-T细胞）增加，可能参与调节肺成纤维细胞增殖及转分化从而促进肺纤维化疾病过程，但其机制尚不明确。本研究通过临床现象、体外细胞实验及动物实验三部分，拟研究衰老T细胞能通过外泌体携带的miRNA作用于肺成纤维细胞，促进肺成纤维细胞增殖分化从而发挥促纤维化作用。研究结果显示免疫衰老现象存在于IPF患者及博来霉素诱导的肺纤维化模型小鼠中。IPF患者外周血中CD3+CD28-T细胞，CD3+CD28-T细胞的培养上清及患者血清都可促进HLF细胞增殖。且HLF细胞的α平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原蛋白（collagen I）的表达明显增加。通过对IPF患者CD3+CD28-T细胞的培养上清中外泌体行miRNA高通量测序，发现miR-320b在IPF患者血浆外泌体中表达明显升高（*P<0.001），miR-483-5p 在IPF患者血浆外泌体中表达升高也有统计学意义（**P=0.0301）。转染miR-320b mimic后发现人肺成纤维细胞明显增殖，α-SMA 、collagen I及collagen III mRNA及蛋白的表达水平均增加，反之，转染miR-320b inhibitor后，人肺成纤维细胞增殖下降，α-SMA 、collagen I及collagen III mRNA及蛋白的表达水平均明显下降。研究认为IPF患者外周血中CD3+CD28-T细胞可能通过外泌体中携带miR-320b促进肺成纤维细胞增殖及转分化从而促进肺纤维化疾病发病过程。