LaminA/C缺失干扰DNA修复介导肺泡上皮细胞衰老参与特发性肺纤维化发病的分子机制研究

Idiopathic pulmonary fibrosis (IPF) is an aging-related disease, and alveolar epithelial cell (AEC) injury plays an important role in the pathogenesis of IPF, DNA damage is a major factor to mediate AEC injury, unrepaired and accumulated DNA damage will lead to AEC senescence. Lamin A/C is an important protein of nuclear membrane skeleton structure, gene mutation in Lamin A/C is a key factor to induce Hutchinson Gilford progeria syndrome (HGPS). Previous study indicate that deficiency of Lamin A/C can result in down-regulation of RAD51, a key protein for DNA homologous recombination (HR) repair, to interfere DNA repair so as to mediate cellular senescence. And from our early experiments, we found that Lamin A/C and RAD51 both decreased in IPF lung tissues, mouse fibrosis lung tissues and senescent AEC induced by bleomycin, whereas DNA damage and AEC senescence significantly increased. According to these previous study and our preliminary experiments, our study contribute to discover whether disorder of DNA repair exist in AEC of IPF and deficiency of Lamin A/C in AEC downregulate RAD51 to interfere DNA damage repair so as to mediate AEC senescence involved in pathogenesis of idiopathic pulmonary fibrosis. In order to validate our hypothesis, gene tools, pharmacological inhibitors and many molecular biological techniques will be applied in our research. This study will provide novel targets for intervention of alveolar epithelial senescence, as well as new ideas for IPF treatment.

特发性肺纤维化（IPF）是一种衰老相关疾病，肺泡上皮细胞（AEC）损伤是IPF发病重要机制，DNA损伤是导致AEC损伤的重要因素，损伤DNA不能及时正确修复将导致AEC衰老，参与IPF发病。Lamin A/C是核膜中核纤层蛋白主要成分，它的突变可导致儿童早老症，研究发现Lamin A/C缺失使DNA损伤同源重组修复关键蛋白RAD51下调，干扰DNA修复，诱导细胞衰老。我们前期研究发现，IPF患者肺组织、博来霉素小鼠肺纤维化和AEC衰老模型中Lamin A/C和RAD51均降低，DNA损伤和衰老标记上调。因此，我们推测Lamin A/C缺失可能通过下调RAD51干扰DNA修复，诱导AEC衰老参与IPF发病。本项目拟在人体、动物、细胞中证实Lamin A/C在AEC衰老中的重要作用，揭示Lamin A/C缺失干扰DNA修复诱导AEC衰老参与IPF发病的分子机制，为治疗IPF提供新靶点。

特发性肺纤维化（IPF）是慢性致肺纤维化的致死性疾病，病情进展快，预后差，目前缺乏有效治疗手段，近年来IPF被认为是一种衰老相关疾病，肺泡上皮细胞（AEC）损伤是IPF发病重要机制，DNA损伤是导致AEC损伤的重要因素，损伤DNA不能及时正确修复将导致AEC衰老，参与IPF发病。Lamin A/C是核膜中核纤层蛋白主要成分，它的突变可导致儿童早老症，研究发现Lamin A/C缺失使DNA损伤同源重组修复关键蛋白RAD51下调，干扰DNA修复，从而诱导细胞衰老。因此，我们猜想Lamin A/C缺失可能通过下调RAD51干扰DNA修复，诱导AEC衰老参与IPF发病。本研究以IPF患者肺组织、博来霉素诱导的小鼠肺纤维化模型、博来霉素诱导的A549细胞衰老模型和原代二型肺泡上皮细胞（AEC2）衰老模型为研究对象，进行免疫组化、免疫印迹、免疫荧光、基因干扰等手段来验证我们的假说。我们的研究结果发现，Lamin A/C和RAD51在IPF患者肺组织、小鼠纤维化肺组织和衰老A549及AEC2细胞中表达均降低，而DNA损伤标记蛋白γH2AX和衰老标记蛋白P16、P21上调，且对IPF患者肺组织和小鼠纤维化肺组织进行免疫组化染色，发现P21主要表达在肺泡上皮细胞，而非成纤维细胞。同时，进行了细胞免疫荧光，发现博来霉素刺激后，细胞核内的RAD51水平降低，γH2AX和P21表达增加，且RAD51、γH2AX与P21细胞核内存在共定位位点。后续将进行针对Lamin A/C、RAD51基因的敲减和过表达，来进一步验证我们的猜想。本项目的科学意义在于进一步证实了IPF是一种衰老相关疾病，且主要是肺泡上皮细胞发生衰老，也证明了DNA损伤是肺泡上皮细胞衰老、肺纤维化的重要发病机制，而Lamin A/C和RAD51可能通过调控DNA损伤修复干扰AEC衰老，是IPF潜在的治疗靶点。