Radiation decreases bronchial epithelial progenitor function as assessed by organoid formation.

IF 5.8 2区医学 Q1 Medicine

Respiratory Research Pub Date : 2025-01-18 DOI:10.1186/s12931-025-03105-z

Merian E Kuipers, Dennis K Ninaber, Krista C J van Doorn-Wink, Annelies M Slats, Pieter S Hiemstra

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引用次数: 0

Abstract

Objective: Radiation-induced lung injury (RILI) is a serious side-effect of radiotherapy for lung cancer, in which effects on the normal lung epithelium may play a key role. Since these effects are incompletely understood, the aim of the present study was to evaluate the effect of ionizing radiation (IR) on cultured well-differentiated primary bronchial epithelial cells (PBEC) with a focus on cytotoxicity, barrier formation, inflammation and epithelial progenitor function.

Materials and methods: PBEC were cultured at the Air-Liquid Interface (ALI-PBEC) to allow mucociliary differentiation. Effect of IR (1, 2, 4, 8 Gy [Gy]) on ALI-PBEC cultures was investigated by lactate dehydrogenase (LDH) release, Trans Epithelial Electrical Resistance (TEER; as a measure of barrier function), qPCR (P21/CDKNA1, MKI67, AEN, E2F1, ATF3) and immunofluorescence staining (γH2Ax-foci). The impact on epithelial progenitor function was assessed by studying organoid formation capacity of irradiated ALI-PBEC at 24 h and 7 days after IR.

Results and discussion: IR increased the number of γH2Ax-foci (marker of double stranded DNA breaks) in ALI-PBEC, but did not affect markers of toxicity (LDH-release or TEER). IR did also not affect mRNA markers for inflammation or epithelial-mesenchymal transition (EMT), but did increase mRNA levels of the cell cycle inhibitor P21/CDKN1A and resulted in downregulation of the proliferation markers MKI67 and E2F1. Finally, IR of ALI-PBEC had a marked effect on organoid formation capacity, which was markedly impaired following IR in a dose-dependent manner.

Conclusion: In conclusion, epithelial progenitor cell function as assessed by organoid formation capacity is strongly reduced by IR and persists for at least 7 days. Despite an effect on organoid formation capacity, DNA breaks, P21/CDKN1A expression and reduced expression of MKI67 and E2F1, this effect was not accompanied by IR-induced cytotoxicity, or an increase in markers of inflammation or EMT. This study indicates that studying the effects of IR on organoid formation is a valid and sensitive tool to study adverse effects of IR on normal lung epithelial cells and could be used as a tool to study RILI.

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通过类器官形成评估，辐射降低支气管上皮祖细胞功能。

目的：放射诱导肺损伤（RILI）是肺癌放疗的严重副作用，其对正常肺上皮的影响可能起关键作用。由于这些影响尚不完全清楚，本研究的目的是评估电离辐射（IR）对培养的良好分化的原代支气管上皮细胞（PBEC）的影响，重点是细胞毒性、屏障形成、炎症和上皮祖细胞功能。材料和方法：在气液界面（ALI-PBEC）下培养PBEC，使其具有粘液纤毛分化。通过乳酸脱氢酶（LDH）释放、上皮反式电阻（TEER）；作为屏障功能的测量),qPCR （P21/CDKNA1, MKI67， AEN, E2F1, ATF3）和免疫荧光染色（γH2Ax-foci）。通过研究ALI-PBEC辐照后24 h和7 d的类器官形成能力来评估对上皮祖细胞功能的影响。结果和讨论：IR增加了ALI-PBEC中γ - h2ax -foci（双链DNA断裂标记）的数量，但不影响毒性标记（ldh释放或TEER）。IR也不影响炎症或上皮间质转化（EMT）的mRNA标记物，但确实增加了细胞周期抑制剂P21/CDKN1A的mRNA水平，并导致增殖标记物MKI67和E2F1的下调。最后，ALI-PBEC的IR对类器官形成能力有显著影响，在IR后，类器官形成能力以剂量依赖的方式明显受损。结论：上皮祖细胞功能（以类器官形成能力评估）在IR下明显降低，且持续至少7天。尽管对类器官形成能力、DNA断裂、P21/CDKN1A表达和MKI67和E2F1表达降低有影响，但这种影响并不伴随着ir诱导的细胞毒性，也不伴随着炎症或EMT标志物的增加。本研究表明，研究IR对类器官形成的影响是研究IR对正常肺上皮细胞不良反应的有效而敏感的工具，可作为研究RILI的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Respiratory Research RESPIRATORY SYSTEM-

CiteScore

9.70

自引率

1.70%

发文量

314

审稿时长

4-8 weeks

期刊介绍： Respiratory Research publishes high-quality clinical and basic research, review and commentary articles on all aspects of respiratory medicine and related diseases. As the leading fully open access journal in the field, Respiratory Research provides an essential resource for pulmonologists, allergists, immunologists and other physicians, researchers, healthcare workers and medical students with worldwide dissemination of articles resulting in high visibility and generating international discussion. Topics of specific interest include asthma, chronic obstructive pulmonary disease, cystic fibrosis, genetics, infectious diseases, interstitial lung diseases, lung development, lung tumors, occupational and environmental factors, pulmonary circulation, pulmonary pharmacology and therapeutics, respiratory immunology, respiratory physiology, and sleep-related respiratory problems.