【作者】 张静；

【导师】 丁楅森；

【作者基本信息】 四川大学 ， 病理学与病理生理学， 2022， 博士

【摘要】 损伤后的器官或组织修复是一个复杂的代谢过程。根据组织的再生能力和炎症反应,结果通常是不完美的,有一定程度的纤维化发生,这是由胶原结缔组织的异常积累造成的。当损伤较轻或非重复性时,创面愈合是有效的,导致ECM成分积累的短暂增加,促进组织结构功能的恢复。然而当损伤是重复性的或严重的,ECM成分继续积累,可导致组织结构的破坏,器官功能障碍,最终器官衰竭。与纤维化相关的常见疾病包括肝硬化、肝炎、非酒精性脂肪性肝炎（NASH）、慢性肾病、心肌梗死、心力衰竭、糖尿病、特发性肺纤维化（IPF）和硬皮病。尽管过去几十年来在器官纤维化领域做出了巨大的努力,并对纤维化的发生和发展取得了相当大的了解,但仍缺乏对纤维化疾病的有效治疗。其中相对有效的是药物治疗和器官移植,药物治疗只能延缓疾病发展的作用,而器官移植受限于器官来源和手术条件及治疗费用。为了更好的研究器官修复与再生中的纤维化反应,我们构建了一系列肝、肺纤维化模型。博来霉素作为抗肿瘤药物,被广泛用于诱导肺纤维化模型。所以我们成功构建了博来霉素诱导的肺纤维化模型。四氯化碳是诱导实验动物产生肝纤维化和肝硬化最广泛使用的药物。所以我们成功构建了四氯化碳诱导的肝纤维化模型。同时我们还构建了小鼠肺再生模型。肺切除术（PNX）会使小鼠的剩余肺叶代偿性生长。PNX模拟了在许多慢性破坏性肺部疾病,对组织损伤的代偿反应可以很容易地量化,这使得PNX成为一个潜在有用的模型,以研究肺再生与修复过程中的细胞作用和分子机制。我们对野生小鼠进行了PNX手术,对小鼠全肺组织进行了单细胞测序,通过分析发现PNX组小鼠的内皮细胞比对照组显著增多,表明内皮细胞在肺再生中发挥重要作用。随后又利用磁珠分离了小鼠肺内皮细胞,进行了单细胞测序,对内皮细胞进行分群发现PNX组小鼠周期性内皮细胞显著增多。而通过进一步分析发现免疫反应相关的cGAS在周期性内皮细胞中高表达。cGAS在肝肺疾病中到底发挥怎样的作用,我们利用cGAS的抑制剂RU.521进行了探究。首先在肺纤维化小鼠造模的同时注射RU.521,进行病理染色,HE和天狼猩红染色发现博来霉素加RU.521组相比单纯博来霉素组肺纤维化加重。肝纤维化小鼠造模的同时注射RU.521,HE、天狼猩红及免疫荧光染色发现四氯化碳加RU.521组相比单纯四氯化碳组纤维化加重。以上结果共同表明抑制cGAS导致肝、肺纤维化加重。为了进一步验证内皮细胞中cGAS在器官修复再生中的作用,我们构建了内皮特异性敲除cGAS的小鼠(cGAS^iΔEC/iΔEC)。对cGAS^+/+小鼠和cGAS^iΔEC/iΔEC小鼠同时进行PNX手术,发现cGAS^iΔEC/iΔEC小鼠肺功能减弱。HE和天狼猩红染色发现cGAS^iΔEC/iΔEC小鼠比对照组小鼠肺纤维化加重。α-SMA和Collagen?免疫荧光染色也发现cGAS^iΔEC/iΔEC小鼠比对照组小鼠肺纤维化严重。EDU染色结果表明cGAS^iΔEC/iΔEC小鼠比对照组小鼠肺内皮细胞增殖减少。对cGAS^+/+小鼠和cGAS^iΔEC/iΔEC小鼠同时进行博来霉素肺纤维化造模,也发现cGAS^iΔEC/iΔEC小鼠比对照组小鼠肺纤维化严重。为了观察肺再生过程中血管新生的情况,我们对肺组织进行了扫描电镜成像,发现cGAS^iΔEC/iΔEC小鼠血管新生增多。同时进行了血管铸型实验,发现肠套叠式血管新生在cGAS^iΔEC/iΔEC小鼠更多的发生,而肠套叠式血管新生是不依赖于细胞增殖发生的血管新生模式,它主要是向血管内部凹陷形成肠套叠支柱,而过多的肠套叠血管生成将对血管造成损伤。以上结果共同说明内皮特异性敲除cGAS后,小鼠肺再生过程中肺纤维化加重,增殖减少,血管受损。为了进一步探究内皮特异性敲除cGAS的对小鼠肺再生影响的机制,我们对cGAS^iΔEC/iΔEC小鼠肺切后进行了单细胞测序。分析发现cGAS^iΔEC/iΔEC组比对照组小鼠内皮细胞显著减少。对小鼠肺内皮细胞进一步分群分析发现cGAS^iΔEC/iΔEC组比对照组周期性内皮细胞显著减少。而cGAS在周期性内皮细胞中高表达。而缺失的周期性内皮细胞与凋亡相关。进一步分析发现cGAS^iΔEC/iΔEC组内皮细胞凋亡比对照组显著增加。我们猜测cGAS通过调控细胞凋亡影响小鼠肺损伤修复。我们通过建立小鼠肝、肺纤维化模型对器官纤维化机制进行研究。通过单细胞测序发现内皮细胞中cGAS在器官修复再生中发挥重要作用,cGAS抑制剂RU.521会导致小鼠肝、肺纤维化加重。同样内皮特异敲除cGAS导致小鼠肝纤维化加重,抑制肺再生及修复。我们猜测cGAS通过调控细胞凋亡影响小鼠肺损伤修复。而具体机制有待进一步研究。更多还原

【Abstract】 Organ or tissue repair after injury is a complex metabolic process.Depending on the regenerative capacity of the tissue and the inflammatory response,the results are often imperfect,with some degree of fibrosis occurring,which is caused by the abnormal accumulation of collagenous connective tissue.Wound healing is efficient when the injury is mild or non-repetitive,resulting in a transient increase in the accumulation of ECM components that promotes the restoration of tissue structure and function.However,when the injury is repetitive or severe,ECM components continue to accumulate,which can lead to disruption of tissue architecture,organ dysfunction,and ultimately organ failure.Common diseases associated with fibrosis include cirrhosis,hepatitis,nonalcoholic steatohepatitis（NASH）,chronic kidney disease,myocardial infarction,heart failure,diabetes,idiopathic pulmonary fibrosis（IPF）,and scleroderma.Despite tremendous efforts in the field of organ fibrosis and considerable understanding of fibrosis initiation and progression over the past few decades,there is still a lack of effective treatments for fibrotic diseases.Among them,drug therapy and organ transplantation are relatively effective.Drug therapy can only delay the development of the disease,while organ transplantation is limited by the source of organs,surgical conditions and treatment costs.In order to study the fibrotic response in organ repair and regeneration,we constructed a series of liver and lung fibrosis models.As an antitumor drug,bleomycin is widely used to induce pulmonary fibrosis models.So we successfully constructed a model of bleomycin-induced pulmonary fibrosis.Carbon tetrachloride is the most widely used drug to induce liver fibrosis and cirrhosis in experimental animals.So we successfully constructed a carbon tetrachloride-induced liver fibrosis model.At the same time,we also constructed a mouse lung regeneration model.Pneumonectomy（PNX）resulted in compensatory growth of the remaining lung lobes in mice.PNX mimics that in many chronic and devastating lung diseases,the compensatory response to tissue damage can be easily quantified,making PNX a potentially useful model to study cellular roles and molecular mechanisms during lung regeneration and repair.We performed PNX surgery on wild mice and single-cell sequencing of the whole lung tissue of the mice.Through analysis,we found that the endothelial cells of the mice in the PNX group were significantly increased compared with the control group,indicating that endothelial cells play an important role in lung regeneration.Subsequently,the mouse lung endothelial cells were isolated by magnetic beads,and single-cell sequencing was performed.Further analysis found that immune response-related cGAS was highly expressed in periodic endothelial cells.What is the role of cGAS in liver and lung diseases,we used cGAS inhibitor RU.521 to explore.First,RU.521 was injected at the same time as pulmonary fibrosis mouse model,Pathological staining,HE and Sirius red staining found that the bleomycin plus RU.521 group had aggravated pulmonary fibrosis compared with the bleomycin group alone.Liver fibrosis mice were injected with RU.521 at the same time,HE,Sirius red and immunofluorescence staining showed that the fibrosis in the carbon tetrachloride plus RU.521 group was aggravated compared with the carbon tetrachloride group alone.These results together indicate that inhibition of cGAS leads to aggravation of liver and lung fibrosis.To further verify the role of cGAS in endothelial cells in organ repair and regeneration,we constructed endothelial-specific cGAS knockout mice(cGAS^iΔEC/iΔEC).Simultaneous PNX surgery was performed on cGAS^+/+mice and cGAS^iΔEC/iΔEC mice,and it was found that lung function was reduced in cGAS^iΔEC/iΔEC mice.HE and Sirius red staining showed that cGAS^iΔEC/iΔEC mice had more pulmonary fibrosis than control mice.α-SMA and Collagen?immunofluorescence staining also found that cGAS^iΔEC/iΔEC mice had more severe pulmonary fibrosis than control mice.The results of EDU staining showed that the proliferation of lung endothelial cells in cGAS^iΔEC/iΔECmice was reduced compared with that in control mice.Bleomycin pulmonary fibrosis modeling was performed on cGAS^+/+mice and cGAS^iΔEC/iΔEC mice at the same time,and it was found that cGAS^iΔEC/iΔEC mice had more severe pulmonary fibrosis than control mice.In order to observe the angiogenesis during lung regeneration,we performed scanning electron microscopy imaging on lung tissue and found that angiogenesis was increased in cGAS^iΔEC/iΔEC mice.At the same time,angiogenesis experiments were carried out,and it was found that intussusception angiogenesis occurred more frequently in cGAS^iΔEC/iΔEC mice,while intussusception angiogenesis was an angiogenesis pattern that did not depend on cell proliferation.Internal depressions form intussusception struts,and excessive intussusception angiogenesis will damage the vessels.These results together indicate that after endothelial-specific knockout of cGAS,pulmonary fibrosis is aggravated,proliferation is decreased,and blood vessels are damaged during lung regeneration in mice.To further explore the mechanism underlying the effect of endothelial-specific knockout of cGAS on mouse lung regeneration,we performed single-cell sequencing in cGAS^iΔEC/iΔEC mice after lung resection.The analysis found that the cGAS^iΔEC/iΔECgroup had significantly fewer endothelial cells than the control group.Further subgroup analysis of mouse lung endothelial cells found that the cGAS^iΔEC/iΔEC group had significantly fewer periodic endothelial cells than the control group.In contrast,cGAS is highly expressed in periodic endothelial cells.The absence of periodic endothelial cells is associated with apoptosis.Further analysis found that the apoptosis of endothelial cells in the cGAS^iΔEC/iΔEC group was significantly higher than that in the control group.We speculate that cGAS affects the repair of mouse lung injury by regulating apoptosis.We studied the mechanism of organ fibrosis by establishing a mouse liver and lung fibrosis model.Through single-cell sequencing,it was found that cGAS in endothelial cells plays an important role in organ repair and regeneration,and the cGAS inhibitor RU.521 will cause aggravation of liver and lung fibrosis in mice.Similar endoterodystic knockout cGAS led to worsening of liver fibrosis in mice,inhibiting lung regeneration and repair.We suspect that cGAS affects lung injury repair in mice by regulating apoptosis.The specific mechanism needs to be further studied..更多还原