巴拉刈誘導小鼠肺部具有幹細胞特性之Clara細胞死亡及其訊息傳遞途徑之研究

The Study for Signal Pathway of Paraquat Induced Cell Death in Mice Pulmonary Clara Cell-Related Stem/Progenitor Cells

黃佳偉(Chia-Wei Huang)

指導教授 ： 彭福佐

共同指導教授 ： 林泰元

國立臺灣大學/醫學院/毒理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01895

摘要

Paraquat(巴拉刈)在1962年首度上市販賣，是目前世界上使用最為廣泛的除草劑之一，經常因誤食進入人體內，於肺部造成大量累積後，會造成嚴重的肺毒性而致死。
由於paraquat的化學結構與動物體內的內生性聚胺（endogenous polyamines）結構類似，因此會經由聚胺吸收系統（polyamine uptake system）被細胞攝入，過去的研究指出，肺部paraquat之累積主要發生在第一型（alveolar Type I）、第二型肺泡上皮細胞（alveolar Type II epithelial cells）與Clara細胞。Paraquat進入細胞之後會被NADPH cytochrome p450 reductase還原成帶單正電之paraquat離子，此一離子態為不穩定之結構，會快速地再度被氧化，進而形成一個氧化還原循環，消耗NADPH，並產生大量的超氧陰離子（O2-）形成自由基攻擊細胞造成傷害。
過去文獻曾經報導第二型肺泡上皮細胞與肺腺癌細胞株A549在曝露paraquat之後會導致細胞內的自由基增加，致使粒腺體的膜電位喪失，釋放出細胞色素C，造成細胞之凋亡；然而在paraquat造成Clara細胞之傷害與其分子機制卻一直沒有相關的研究，同時Clara細胞富含cytochrome P450 monooxygenase與NADPH cytochrome p450 reductase，對於肺部外來物質代謝上扮演著重要的角色，因此引發了本實驗的動機來探討paraqaut對於Clara細胞造成的傷害及影響。然而目前沒有容易取得且被認定之Clara細胞株，實驗乃利用林泰元博士於2006年美國國家科學院院刊（PNAS）上發表的實驗方法，將乳鼠肺臟內的Clara cell-related stem/progenitor cells取出做原代培養，來探討paraquat對Clara cell-related stem/progenitor cells毒性之研究。
本實驗以下列所述之研究方法進行：（1）鑑定CCSP與Oct-4之蛋白表現來確認所培養之細胞為Clara cell-related stem/progenitor cells。（2）接著將細胞處理不同濃度、不同時間之paraquat，利用觀察細胞形態的改變、測量LDH釋放量、TUNEL螢光染色以及流式細胞儀之分析，來評估在不同條件處理下，paraquat所引發Clara cell-related stem/progenitor cells的傷害模式為何。（3）鑑定Clara cell-related stem/progenitor cells細胞膜上ABCB1轉運蛋白之表現。（4）測量曝露paraquat後，細胞內caspase活性與細胞培養液中FasL濃度之改變。（5）以FasL的抗體進行對細胞培養液中之FasL進行免疫中和反應，再利用TUNEL螢光染色與流式細胞儀分析其對細胞凋亡之影響。（6）測量曝露paraquat後，細胞所分泌細胞激素之變化。（7）利用不同濃度之paraquat誘導小鼠之肺部傷害，並於傷害造成後移殖具有GFP表現能力之Clara cell-related stem/progenitor cells進入小鼠體內，利用免疫螢光染色與螢光顯微鏡觀察其更新與修復之情形。
目前的實驗結果顯示，曝露高濃度之paraquat會導致細胞壞死，而低濃度之paraquat會誘導Clara cell-related stem/progenitor cells與mesenchymal stroma cells產生細胞凋亡，其中Clara cell-related stem/progenitor cells對paraquat具有更高之耐受性，其耐受性可能是來自於Clara cell-related stem/progenitor cells細胞膜上之ABCB1轉運蛋白，ABCB1能夠將細胞內的paraquat排出胞外而減少其造成之傷害。此外分析caspase活性改變的結果，發現paraquat會誘導caspase 2、3、4、8、9與caspase 10之活性上升，並降低caspase 1之活性，顯示paraquat所誘導之細胞凋亡會經由內在調控與外在調控兩條不同的途徑。而透過測量FasL之濃度，發現曝露paraquat會使細胞培養液中FasL的表現量上升，進一步利用FasL之抗體對細胞培養液中的FasL進行免疫中和反應，結果顯示能夠有效降低paraquat所誘發之細胞凋亡。另外，測量細胞曝露paraquat後細胞激素分泌之改變，亦發現FasL之表現量會隨著時間而上升；另外在與發炎相關的細胞激素中，亦發現IFN-γ、IL-1β、IL-3、IL-5、IL-6、IL-17、MIP1-γ、MIP3-γ、Eotaxin-2、FKN皆有上升之趨勢；在與肺部纖維化相關之細胞激素方面，可以發現MMP-2的上升與pro-MMP9的下降，同時也發現細胞生長因子GCSF、IGF-1、IGFBP-2、IGFBP-5、IGFBP-6、SCF、SDF-1α、VEGF與可溶性受體sTNFRI之表現量皆有上升。在動物實驗之結果顯示，當paraquat誘導小鼠肺部產生傷害後，移殖進入小鼠體內具有GFP表現能力的Clara cell-related stem/progenitor cells會回歸至支氣管上皮的位置。
　　綜合以上結果，本研究證實paraquat會透過內在調控與外在調控兩條不同之訊息傳導途徑來誘導Clara cell-related stem/progenitor cells與mesenchymal stroma cells之細胞凋亡，而FasL可能於paraquat誘導外在途徑調控之細胞凋亡中扮演重要的角色，同時paraquat亦會刺激Clara cell-related stem/progenitor cells與mesenchymal stroma cells分泌凋亡、發炎與肺部纖維化等相關細胞激素。此外，本研究亦發現具有分化能力之Clara cell-related stem/progenitor cells可能參與paraquat造成肺部傷害後的修補、保護作用。

關鍵字

巴拉刈 ； 肺 ； 幹細胞 ； 源祖細胞 ； 細胞凋亡 ； 氧化壓力

並列摘要

Paraquat (PQ), the bipyridyl herbicide, is a strong pneumotoxicant for lung epithelial cells. PQ was accumulated in the cells through a polyamine uptake system and induced redox cycling, leading to oxidative stress-related damage and NADPH depletion. Previous studies showed that paraquat exposure result in the increase of reactive oxygen species, loss of mitochondrial membrane potential, release of cytochrome c, and cell apoptosis. In lung tissue, it was well known that the Clara cells play important role in the metabolism of xenobiotic in the lung, because they have abundant cytochrome P450 monooxygenase and NADPH cytochrome p450 reductase. However, the molecular mechanisms involved in PQ-induced Clara cells injury remains unclear.
In this work, we set a primary culture of Clara cell-related stem/progenitor cells surrounding with mesenchymal stroma cells to examine the PQ-induced cell death. Different concentration of PQ and time couse treatments were applied to culture system.The LDH release assay showed the high concentration of paraquat cause cell necrosis in the whole culture system. In low concentrations of PQ, the analysis of DNA fragmentation with TUNEL staining and Flow Cytometry showed the PQ-induced cell apoptosis in Clara cell-related stem/progenitor cells and mesenchymal stroma cells, and it was great interesting that the Clara cell-related stem/progenitor cells were more resistant than mesenchymal stroma cells to the paraquat. The resistance could be attributed to the ABCB1 transporter located at the cell membrane of Clara cell-related stem/progenitor cells. Previous studies have shown ABCB1 could export paraquat out of the cell and attenuate the PQ toxicity.
The study also indicated paraquat exposure increased caspase 2, 3, 4, 8, 9, and caspase 10 activities but decreased caspase 1 activity. Resulting from the variation of caspase activity, the paraquat-induced cell apoptosis were both through intrinsic and extrinsic apoptotic pathway. Paraquat exposure also increased the concentration of FasL in the cultured medium, and the antibody neutralization of FasL could prevent paraquat-induced cell apoptosis. By cytokine antibody array analysis, paraquat exposure also increased the expression of cytokines, growth factors and soluble receptors, such as FasL, IFN-γ, IL-1β, IL-3, IL-5, IL-6, IL-17, MIP1-γ, MIP3-γ, Eotaxin-2, FKN, MMP-2, GCSF, IGF-1, IGFBP-2, IGFBP-5, IGFBP-6, SCF, SDF-1α, VEGF, and sTNFRI in the cultured medium.
In animal model, the Clara cell-related stem/progenitor cells and mesenchymal stroma cell were transplanted into mice via intravenous injection by tail vein after paraquat-induced lung injury, and the cells were homing back majority to the bronchoalveolar junction where the Clara cells were usually located.
Taken together, the current study showed that paraquat activates the intrinsic and extrinsic apoptotic pathways of Clara cell-related stem/progenitor cells and mesenchymal stroma cell, and the FasL might play an important role in induction of extrinsic apoptotic pathways. Paraquat exposure also induced the variation of cytokines expression, and might result in inflammation and fibrosis of lung. In animal model, our data also suggested that the Clara cell-related stem/progenitor cells might contribute the regenerative mechanism of airway epitheliuml.

並列關鍵字

Paraquat ； lung ； Clara cell ； stem cell ； progenitor cell ； apoptosis ； oxidative stress

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