高氧誘導肺臟幹細胞損傷之機制探討

To study the mechanism of hyperoxia-induced injury of pulmonary stem/progenitor cells.

余柏毅(Bo-Yi Yu)

指導教授 ： 林泰元

國立臺灣大學/醫學院/藥理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602746

摘要

高純度氧氣療法已被納入現今許多疾病的治療準則之中，但不可否認地它伴隨的副作用如結構損傷或發炎反應仍然遍及各種組織器官，而其中肺臟的損傷尤甚。目前關於高氧造成肺部損傷之研究多數仍集中在第二型肺泡細胞及肺臟內皮細胞的損傷模式等等，但有關於高純度氧氣如何對第一型肺泡細胞及其幹細胞造成影響，而此影響又是如何參與在肺部損傷的病理機制之中，都還尚未被廣泛討論。為了釐清高氧誘導之肺部損傷的機制並找到合適的治療方法，我們將在之前的研究中建立好的類第一型肺泡細胞之分化模型暴露於高純度氧氣的環境中，並且發現隨著老鼠肺部幹細胞分化成類第一型肺泡細胞之過程中，細胞對於高純度氧氣的抗性也會隨之上升。接著我們透過生物資訊工具分析肺部幹細胞分化過程之全基因表現資料推測第三型穀胱甘肽過氧化物酶(GPx 3)可能於此抗性中扮演關鍵的角色，因此又進行了後續的即時聚合酶鏈鎖式反應、西方墨點法，及免疫螢光細胞染色法證實了其在老鼠肺部幹細胞分化中的表現量差異。而在以藥物抑制第三型穀胱甘肽過氧化物酶的酵素作用之實驗中也再度得證了其在已分化成熟的老鼠肺部幹細胞中抵禦高純度氧氣之重要性。綜合以上實驗的結果，我們合理地推測隨著老鼠肺部幹細胞分化而增加其表現量的第三型穀胱甘肽過氧化物酶對於其獲得的抵禦高純度氧氣損傷之抗性有高度的相關性。

關鍵字

高氧 ； 肺臟幹細胞 ； 自由基 ； 第三型穀胱甘?過氧化物?

並列摘要

Hyperoxia-based treatment has been approved for many disease indications from long times ago, but its adverse effect such as structural damages and inflammation of tissues, especially lungs, still cannot be ignored. Numerous researches focused on endothelial cells and type 2 pneumocytes of lungs, but the role of type 1 pneumocytes and its stem cells in pathological events of hyperoxia-induced lung injuries was little discussed. To deeply understand the pathology of hyperoxia-induced lung injuries and search for its potential therapies, we applied hyperoxia treatments to a type 1-like pneumocytes differentiation model of mouse lung stem/progenitor cells (mPSCs) established in our lab in previous studies and discovered a growing resistance accompanied with differentiation. Through bioinformatics tools to analyze the whole genome gene expression data of mPSCs, we supposed the potential role of glutathione peroxidase 3 (GPx3) in the mechanism of acquired hyperoxia-resistance of mPSCs. Also, the differential expression of GPx3 was validated by the results of real-time qPCR, western blot, and immunocytochemistry and inhibition of GPx enzyme activities in cell proliferation and intracellular ROS accumulation assays further confirmed its indispensability in anti-oxidant defensing system of mPSCs. Based on the evidence of our experiments, we supposed that the growing expression of GPx3
provided the resistance to hyperoxia environment for well-differentiated mPSCs.

並列關鍵字

Hyperoxia ； lung stem cell ； ROS ； GPX3

參考文獻

1. Ahmed, M. N., Suliman, H. B., Folz, R. J., Nozik-Grayck, E., Golson, M. L., Mason, S. N., Auten, R. L. (2003). Extracellular superoxide dismutase protects lung development in hyperoxia-exposed newborn mice. Am J Respir Crit Care Med, 167(3), 400-405. doi:10.1164/rccm.200202-108OC

2. Alejandre-Alcazar, M. A., Kwapiszewska, G., Reiss, I., Amarie, O. V., Marsh, L. M., Sevilla-Perez, J., . . . Morty, R. E. (2007). Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol, 292(2), L537-549. doi:10.1152/ajplung.00050.2006

3. Angara, S., Syed, M., Das, P., Janer, C., Pryhuber, G., Rahman, A., . . . Bhandari, V. (2016). Inhibition of RPTOR Prevents Hyperoxia-induced Lung Injury by Enhancing Autophagy and Reducing Apoptosis in Neonatal Mice. Am J Respir Cell Mol Biol. doi:10.1165/rcmb.2015-0349OC

4. Arner, E. S., Holmgren, A. (2000). Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem, 267(20), 6102-6109.

5. Arthur, J. R. (2000). The glutathione peroxidases. Cell Mol Life Sci, 57(13-14), 1825-1835.