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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 彭福佐(Fu-Chuo Peng) | |
dc.contributor.author | Chia-Yuan Tang | en |
dc.contributor.author | 湯佳元 | zh_TW |
dc.date.accessioned | 2021-06-15T01:12:59Z | - |
dc.date.available | 2016-10-07 | |
dc.date.copyright | 2011-10-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42387 | - |
dc.description.abstract | 巴拉刈 (Paraquat, PQ) 是一種廣泛使用的除草劑,但卻常因為誤食而造成中毒死亡。由於paraquat的化學結構與內生性多胺 (endogenous polyamine)相似,所以可以藉由肺臟的第一型和第二型肺泡上皮細胞 (alveolar type I and type II epithelial cells)及Clara細胞的多胺運輸系統 (polyamine uptake system, PUS) 運輸進到細胞內,因此當paraquat進到人體後,經由血液循環到各組織器官,尤其主要累積在肺臟。而當paraquat進入細胞後,隨即會進行氧化還原循環(redox cycling),導致NADPH的氧化以及自由基的產生,進而造成細胞的傷害。
實驗室由小鼠肺臟中培養出肺臟幹源細胞,這群細胞具有octamer-binding transcription factor 4 (Oct-4)、stage-specific embryonic antigen-1 stem cell antigen 1 (Sca-1)等幹細胞標誌蛋白的表現。此外,細胞也會表現CCSP以及細胞色素P450酵素。然而paraquat對於小鼠肺臟幹源細胞造成的傷害及影響並不清楚,在先前的研究中觀察到特定濃度paraquat會誘導stroma cells走向細胞凋亡,其中epithelial colony cells具有更高之耐受性。又目前對於paraquat如何排出胞外的研究仍為數不多,主要著重在ABCB1轉運蛋白的探討。因此,本篇目的在於探討ABCB1是否在paraquat造成細胞死亡的機制中是否扮演重要的角色。 實驗首先以不同濃度paraquat處理小鼠肺臟幹源細胞,並與不同肺部細胞株 (BEAS-2B, A549, MLE-15) 進行存活率之比較,實驗結果顯示,小鼠肺臟幹源細胞相較於其他肺部細胞株較具有耐受性,而小鼠肺臟幹源細胞中,epithelial colony cells相較於stroma cells具有更高的耐受性。接著實驗以免疫螢光染色進行ABCB1的分析,並利用verapamil 抑制ABCB1 轉運蛋白的作用來探討細胞存活率以及胞內ROS是否受到影響。結果發現,抑制ABCB1 轉運蛋白的作用會加劇paraquat造成小鼠肺臟幹源細胞的傷害,並且會增加胞內ROS的產生量。此外,實驗中也利用RT-PCR和qPCR去探討ABCB1 mRNA表現,結果發現隨著paraquat處理時間增加,ABCB1 mRNA的表現量也會增加。 此外,細胞受到paraquat刺激後,會造成細胞凋亡的發生。而先前研究觀察到隨著paraquat處理濃度的增加,培養液中測得的FasL也會增加。因此,實驗進一步去探討FasL表現的位置,以RT-PCR和western blot去偵測FasL及其受體Fas的表現量,結果顯示FasL只表現在stroma cells,而隨著暴露時間的增加,Fas及FasL的表現也會增加。最後,為了探討外在途徑調控之細胞凋亡機制是否有被活化,實驗測量pro-caspase 8和pro-caspase 3的表現量,結果發現,隨著暴露時間的增加,pro-caspase 8和pro-caspase 3的表現量均減少,顯示細胞凋亡之外在途徑已被活化。 綜合上述結果,本研究推論肺臟幹源細胞受到paraquat刺激下,會藉由ABCB1轉運蛋白將paraquat排出胞內達到保護的作用;雖然ABCB1表現會被刺激而增加,但在paraquat刺激的過程中,外在途徑調控之細胞凋亡機制會被活化,因此仍會造成細胞走向死亡。 | zh_TW |
dc.description.abstract | Paraquat (PQ) is one of the most widely used herbicides in the world, and it is the well-known pneumotoxicant. Paraquat accumulates mainly in the lungs through polyamine uptake system and undergoes a process of redox-cycling, which leads to reactive oxygen species (ROS) production at the expense of NADPH.
We have set a primary culture of mouse pulmonary stem/progenitor cells (mPSCs) which includes epithelial colony cells and surrounding stroma cells in theculture system. However, the toxicity of cellular response to PQ in mPSCs was not elucidated. According to our previous data, we found stroma cells were more sensitive to PQ than epithelial colony cells. This difference between two cells may be explained by the expression of ABCB1 transporter. Hence, the aim of this study is to investigate the role of ABCB1 in PQ-induced cell death in mPSCs. First, we examined and compared cell viability of PQ on mPSCs and three lung cell lines, such as BEAS-2B, A549 and MLE-15 cells. By MTT assay, it showed mPSCs were more resistant to PQ among these cells. Then, we evaluated the relationship between ABCB1 transporter and mPSC PQ’s cell viability. The results indicared that ABCB1 was mainly expressed on the membrane of epithelial colony cells, and inhibition of ABCB1 transporter by verapamil caused more intracellular ROS production when administrated with paraquat, which leaded to lower cell viability. Our previous study also showed FasL expression was increased in response to paraquat-induced cell apoptosis in mPSCs. Therefore, we investigated whether the FasL was located in epithelial colony cells or stroma cells. The results showed that FasL was expressed in the surrounding stroma cells but not epithelial colony cells. The expression of Fas and FasL in stroma cells was increased in a time-dependent manner. To verify whether Fas/FasL pathway was activated in stroma cells, the expression of pro-caspase 8 and pro-caspase 3 was investigated. It indicated that the extrinsic apoptotic pathway was activated despite the induction of ABCB1 mRNA expression in stroma cells. Taken together, the current study showed that ROS was involved in PQ-induced mPCSs cell death, and ABCB1 may export PQ out of cells and attenuated the PQ toxicity. Furthemore, FasL might play an important in induction of extrinsic apoptotic pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:12:59Z (GMT). No. of bitstreams: 1 ntu-100-R98447009-1.pdf: 1679756 bytes, checksum: 7c58c51856819737f170ee7dd5b8e682 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Contents
Abbreviations ……………………………………………………………… iv List of figures and tables ……………………………………………………… v 中文摘要 …………………………………………………………………… vi Abstract ………………………………………………………………………… viii Chapter 1 Introduction 1.1. History, Use and Poisoning cases of Paraquat…..……………….……………… 2 1.2. Absorption and Distribution of Paraquat…..……………….…………………… 3 1.3. Pulmonary Stem/Progenitor Cell……………………………………………… 11 1.4. Biochemical Mechanism of Paraquat Toxicity ……………………………… 6 1.4.1. Mechanism of Toxicity………………………………………………… 6 1.4.2. Biochemical consequences of the redox cycling process.………………… 6 1.5.Paraquat-Induced Cell Apoptosis ……………………………………………… 8 1.5.1. The apoptotic pathway ………………………………………………… 8 1.5.2. The intrinsic pathway of apoptosis……………………………………… 8 1.5.3. The extrinsic pathway of apoptosis……………………………………… 9 1.5.4. PQ-induced extrinsic pathway of apoptosis……………………………… 10 1.6. Elimination of Paraquat………………………………………………………… 10 1.7. Motivations and aims………………………………………………………… 12 Chapter 2 Materials and methods 2.1. Cell Culture Expansion ……………………………………………………… 15 2.1.1. Primary culture of mPSCs (mouse pulmonary stem/progenitor cells) … 15 2.1.2. Culture of cell lines ………………………………………………… 16 2.2. Colorimetric MTT (tetrazolium) assay ……………………………………… 16 2.3. Immunofluorescence staining ……………………………………………… 17 2.4. In situ detection of apoptosis ……………………………………………… 18 2.5. RNA extraction and quantification ……………………………………… 18 2.6. Reverse transcription and real time-quantitative PCR ……………………… 19 2.7. Measurement of reactive oxygen species (ROS) production………………… 20 2.8. Cell lysate collection………………………………………………………… 21 2.9. Western blot………………………………………………………………… 21 2.10. Statistical Analysis ………………………………………………………… 22 Chapter 3 Results 3.1. Effect of Paraquat on Viability of Lung Cell Lines and Mouse Pulmonary Stem/Progenitor Cells (mPSCs) …………………………………………… 25 3.2. Paraquat-Induced Morphological Alteration and Cell Apoptosis in mPSCs … 26 3.3. Expression and Effects of ABCB1 Transporter in mPSCs after Paraquat Treatment………………………….………………………………………… 28 3.5. Relation between Reactive Oxygen Species (ROS) production and ABCB1 Transporter in mPSC after Paraquat Treatment..……………………………… 30 3.6. Activation of Extrinsic Apoptotic Pathway in Surrounding Stroma Cells after Paraquat Treatment…………………………………………………………… 31 Chapter 4 Discussion 4.1. The detoxification ability in mPSCs........................ ....................................... 35 4.1.1. The role of ABCB1 in PQ detoxification………………………..……… 35 4.1.2. Other detoxification abilities in mPSCs………………………………… 36 4.2. Induction of Fas and FasL by paraquat.....………………………………......... 37 4.3. The apoptotic pathways involved in paraquat-induced toxicity...................... 38 4.4. Future work...……………………………....... ………………….......……......... 40 Chapter 5 Conclusion……....................................................................................... 41 Chapter 6 References …………............................................................................... 43 Fgures and Tables .................................................................................................... 54 | |
dc.language.iso | en | |
dc.title | 巴拉刈中毒於小鼠肺臟幹源細胞解毒機制之探討 | zh_TW |
dc.title | The Study of Detoxification Mechanism in Mouse
Pulmonary Stem/Progenitor Cells for Paraquat Intoxication | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 林泰元(Thai-Yen Ling) | |
dc.contributor.oralexamcommittee | 林國煌(Kuo-Huang Lin),陳耀昌(Yao-Chang Chen),曹伯年(Po-Nien Tsao) | |
dc.subject.keyword | 幹細胞,巴拉刈,ABCB1運輸蛋白,細胞凋亡, | zh_TW |
dc.subject.keyword | pulmonary stem cell,paraquat,apoptosis,ABCB,ROS,Fas ligand, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-16 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 毒理學研究所 | zh_TW |
顯示於系所單位: | 毒理學研究所 |
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