| 摘要: | 背景：低氧是許多病理性疾病的主要特徵，促進器官纖維化。儘管胎兒對氧的需求低於出生後，但子宮血流和母親提供的氧氣仍會影響胎兒。氧的減少可能導致早產和神經系統損傷。我們的研究旨在通過Hippo通路研究缺氧對分枝形態發生的影響。
材料與方法：從懷孕11.5胚胎日ICR小鼠中解剖取得胎兒肺。離體肺在正常氧氣濃度或缺氧環境（1％O2）下培養。通過每24小時計算分枝肺芽數量和肺部面積來分析肺分枝形態發生，共計3天。我們對曝露3天的小鼠胚胎全肺進行RNA測序分析。正常的人類胚胎肺成纖維細胞IMR-90細胞在常氧或缺氧環境下培養24小時。利用上清液測定了乳酸脫氫酵素（LDH）和白細胞介素6（IL-6）。使用西方墨點法確定YAP、TAZ、SOX2、SOX9、FGF10、FGFR2和SIRT1的蛋白表達。免疫螢光染色觀察胎兒肺切片上YAP、TAZ和SOX2的表達。
結果：我們觀察到在缺氧環境中，離體小鼠胎兒肺的分枝形態發育被抑制。缺氧組的末端支氣管數量和肺面積顯著低於對照組（p<0.05）72小時後。此外，胎兒肺和IMR-90細胞的缺氧組中LDH和IL-6增加。西方墨點法的結果表明，在缺氧暴露下，YAP和TAZ的磷酸化增加。SOX2在缺氧組的胚胎肺和IMR-90細胞中顯著下降（p<0.05）。免疫螢光染色顯示，YAP+和SOX2+的表達在暴露於缺氧後的離體胚胎肺上Fibronectin+細胞中減少。另一方面，FGF10在IMR-90細胞受缺氧暴露後過度表達（p<0.05）。小鼠胚胎肺的RNA-測序表明，缺氧胚胎肺中“子宮內胚胎發育”和“對生長因子刺激的反應”與對照組相比減少。
結論：我們的研究結果表明，缺氧環境會減弱胎兒肺的分枝形態發育。此外，缺氧組的細胞毒性和發炎反應增加。此外，YAP / TAZ和下游信號分子可能受早期肺發育期間缺氧的影響，進而導致發育延遲和細胞衰老調控減少。
Introduction
Hypoxia is a prominent feature of many pathological diseases, promoting fibrosis of organs. Although the demand for oxygen in the fetus is lower than after birth, uterine blood flow and maternal oxygen supply still affect the fetus, and the reduction in oxygen can lead to prematurity and damage of nervous system. The aim of our study is to investigate the effects of hypoxia on branching morphogenesis via Hippo signaling pathway.
Materials and methods
Fetal lungs were dissected from 11.5 embryonic days ICR pregnant mice. The ex vivo lungs were cultured under normoxia or hypoxia environment (1% O2). The lung branching morphogenesis was measured by calculating branching lung buds and lung area every 24 hours for 3 days. We performed RNA sequencing analysis on mouse fetal whole lungs after 3 days of exposure. Normal human fetal lung fibroblast IMR-90 cells were cultured under normoxia or hypoxia environment for 24 hours. Biochemical analysis including Sulforhodamine B (SRB), lactate dehydrogenase (LDH) assay, and Interleukin 6 (IL-6) were measured in supernatants. Expression of YAP, TAZ, SOX2, SOX9, FGF10, FGFR2, and SIRT1 was determined using western blot. Immunofluorescence was utilized for observing YAP, TAZ, and SOX2 expression on mouse fetal lung slices.
Results
We observed that in hypoxic environment, branching morphogenesis of ex vivo mouse fetal lungs were inhibited. The number of terminal bronchioles and lung area in the hypoxic environment were significantly lower than that of the control group after 72 hours (p<0.05). Besides, LDH and IL-6 were increased in hypoxia group of fetal lungs and IMR-90 cells. The results of western blot showed that phosphorylation of YAP and TAZ increased under hypoxic exposure. SOX2 significantly decreased in hypoxia group of fetal lungs and IMR-90 cells (p<0.05). Immunofluorescence showed the expression of YAP+ and SOX2+ on Fibronectin+ cells of ex vivo fetal lung decreased after exposure to hypoxia. On the other hand, FGF10 over-expressed in IMR-90 cells after exposure of hypoxia (p<0.05). Mouse fetal lungs of RNA-sequencing demonstrated “in utero embryonic development” and “response to growth factor stimulus” were decreased in hypoxia fetal lung compared to control.
Conclusion
Our findings suggested that hypoxia condition may have the potential to attenuate fetal lungs’ branching morphogenesis. Furthermore, cell cytotoxicity and inflammatory reaction increased in hypoxia group. Also, YAP/TAZ and downstream signaling molecules may be affected by hypoxia during early lung development, which could result in delayed development and reduced regulation of cellular senescence. |
