研究細胞治療對巴拉刈引發小鼠急性呼吸窘迫症之修復能力及機制的探討

The Study on Regeneration and Regulatory Mechanism of Cell Therapy for Paraquat-Induced Acute Respiratory Distress Syndrome in Mice

陳駿傑(Chun-Chieh Chen)

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(2010年)

https://doi.org/10.6342/NTU.2010.02215

摘要

巴拉刈 (paraquat, PQ)，化學名稱為1,1’-dimethyl-4,4’-bipyridylium dichloride，分子式為C12H14Cl2N2，在農藥分類上被歸類為四級胺類 (quaternary ammonium)除草劑，已經在全球超過一百二十個已開發或開發中國家登記上市，是目前世界上使用最廣泛的除草劑之一。由於paraquat的化學結構與內生性多胺 (endogenous polyamine)相似，因此可透過多胺運輸系統 (polyamine uptake system, PUS)運輸進到細胞內。此類的運輸系統主要存在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，因此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。當paraquat進到肺臟細胞內，會進行氧化還原循環 (redox cycling)，消耗大量NADPH，產生超氧化物 (superoxide anion, O2.-)等自由基，導致肺部的傷害。在一般情況下，paraquat造成急毒性大部分是藉由口服誤食。根據2001到2002年的統計資料顯示，在台灣所有農藥中毒事件中，paraquat中毒佔了18.9 %，造成的死亡率為72.1 %。
過去文獻顯示，高劑量paraquat中毒後，會引發多重器官衰竭，其中包括急性呼吸窘迫症 (acute respiratory distress syndrome, ARDS)。在paraquat中毒後，於一到三天內攻擊肺臟之alveolar type I及type II細胞，破壞alveolar type I細胞形成的屏障，以及降低alveolar type II細胞對體液再吸收的能力，使得體液進到肺泡空腔，導致肺臟水腫，並造成肺臟順應性下降，使肺臟無法進行有效的氣體交換。此外，alveolar type I細胞形成的屏障被破壞後，也會導致發炎細胞浸潤到肺臟，其中最主要的細胞類型為嗜中性球 (neutrophils)。而paraquat中毒引起的發炎反應與其死亡率有關，因此可藉由減少發炎反應提高中毒後之存活率。
傳統的治療方法包括催吐 (induction of emesis)，洗胃 (gastric lavage)，血液灌流 (charcoal haemoperfusion)及糖質皮質固醇類 (glucocorticoid)藥物治療，但這些治療往往無法有效提高存活率，即使患者存活下來，其肺臟功能也會受到影響。近年來，有許多研究指出間葉幹細胞 (mesenchymal stem cells, MSCs)具有抑制發炎的功能，且利用間葉幹細胞進行細胞治療在其他疾病的研究中也可見到成效，包括神經退化或心肌梗塞等疾病。因此，從2005年起有越來越多研究探討細胞治療是否可運用在治療肺部病變。然而，在間葉幹細胞培養的過程中，會在培養液當中加入動物血清 (serum)，但當運用在細胞治療時，血清可能會附著在間葉幹細胞中，對於治療的宿主可能產生嚴重的免疫反應。因此，實驗中利用無血清培養的人類絨毛膜間葉細胞 (human chorion-derived mesenchymal cells, CMCs)對於paraquat中毒的小鼠進行細胞治療，觀察是否能減少傷害並提高其存活率，接著探討其中的調節機制。
為了模擬paraquat所造成的急性呼吸窘迫症，實驗首先利用六週大雌性ICR小鼠以口服方式餵食480 mg/kg BW劑量的paraquat。由組織切片中可以發現，此劑量的paraquat會造成小鼠肺臟充血，肺泡壁增厚及發炎細胞浸潤。此外，也會造成ICR小鼠的肺部功能受到顯著的影響，包括肺臟順應性 (pulmonary compliance)，肺臟彈性 (pulmonary elastance)及肺臟組織阻力 (lung tissue resistance)。而在利用人類絨毛膜間葉細胞進行細胞治療後，可將ICR小鼠存活率由8 %提高至35 %，但利用人類支氣管上皮細胞 (Beas2B cells)及dexamethasone (DEX)進行治療皆無法有效提高存活率。此外，利用人類絨毛膜間葉細對處理過paraquat之C57BL/6小鼠進行細胞治療，也可提高其存活率。實驗結果也顯示細胞治療可改善paraquat對小鼠引起的肺臟組織傷害，並減少肺臟功能的破壞，且在治療後第六天可使肺臟功能回復到與控制組相似。同時，進行細胞治療後，利用螢光免疫染色標定人類細胞，可在ICR小鼠的肺臟組織中發現人類絨毛膜間葉細胞，顯示進行細胞治療的人類絨毛膜間葉細胞可循環到受傷的肺臟組織，並進行調控。
接著，藉由肺臟功能變化，肺臟的發炎反應及釋放之細胞激素 (cytokine)的改變來探討細胞治療對於小鼠在paraquat處理後的影響。由肺功能變化來看，細胞治療可以在第二天及第三天有效減少肺臟組織阻力，且在第三天有效提高肺臟順應性。ICR小鼠在paraquat處理後，會造成嗜中性球逐漸浸潤到肺泡空腔及肺臟組織，也會使和嗜中性球浸潤及活化相關的細胞激素濃度增加。然而，細胞治療可以有效減少這些細胞激素的上升，並且降低浸潤到肺泡空腔及肺臟組織的嗜中性球。
綜合以上結論，實驗結果顯示在高劑量480 mg/kg BW paraquat處理後的ICR小鼠，以人類絨毛膜間葉細胞進行細胞治療，可以減少小鼠肺臟傷害，回復肺臟功能並且提高存活率。而這樣的回復現象，或許是藉由減少與嗜中性球浸潤及活化相關之細胞激素的濃度，以及減少嗜中性球浸潤到肺泡空腔及肺臟組織。

關鍵字

巴拉刈 ； 急性呼吸窘迫症 ； 肺部功能 ； 人類絨毛膜間葉細胞 ； 細胞治療 ； 嗜中性球浸潤

並列摘要

Paraquat (PQ) is herbicide and also a potent pneumotoxicant. Paraquat mainly accumulates in the lungs through polyamine uptake system and undergoes a process of redox-cycling and leads to reactive oxygen species (ROS) production at the expense of NADPH. This alters the normal cell functions and causes lung toxicity. Paraquat poisoning is the most common cause of fulminated and fatal herbicide intoxication. From 2001 to 2002, paraquat intoxication contributed to 18.9 % of all pesticide poisonings in Taiwan.
Paraquat intoxication evokes sudden reproducible lung damage which mimics the clinical course of the patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In the early stages, loss of the alveolar capillary barrier integrity develops and leads to formation of pulmonary edema, which contributes to impairment of gas exchange and loss of compliance. Concurrently, there is also a massive influx of inflammatory cells into the lungs. The severe inflammation may play a vital role in high mortality at the period of paraquat poisoning, and the survival may be improved by attenuating lung inflammation.
The conventional clinical therapies for paraquat intoxication are insufficient for the patients with fulminant paraquat poisoning. Even the patients survived, they still had restrictive pulmonary dysfunction. Recently, mesenchymal stem cells (MSCs) have been reported to have the ability to attenuate tissue inflammation, reduce lung injury and improve survival. MSCs are currently cultured and expanded in the presence of serum. However, the use of animal serums may be incorporated by MSCs during culture procedures and cause immune reactions in the host after administration. Therefore, this study evaluated the treatment efficacy of serum-free-cultured human chorion-derived mesenchymal cells (CMCs) therapy in mice with acute fulminant paraquat poisoning, and then investigated the consequences and effects.
In animal model, to mimic ALI/ARDS, female ICR mice (6 weeks) were orally administered with 480 mg/kg BW of paraquat. Histopathologically, the lungs showed pulmonary hemorrhage, thickening of alveolar septum and inflammatory cells infiltration. Further, respiratory mechanics assessment also revealed significant difference in pulmonary compliance, pulmonary elastance and lung tissue resistance compared to control group. Due to the lack of effective treatment in acute fulminant paraquat poisoning, the experiment co-treated serum-free-cultured human CMCs to mice to evaluate the treatment efficiency. The results showed that the survival rate of ICR mice with acute fulminant paraquat poisoning increased from 8 % to 35 % after CMCs co-treatment, but Beas2B cells and dexamethasone (DEX) co-treatment did not recover the survival rate. Similarly, CMCs co-treatment could also apply to other strain, such as C57BL/6 mice. Histopathologically, CMCs co-treatment recovered the paraquat-induced lung injury, and the lung architecture was similar to control group. The pulmonary functions were also restored by CMCs co-treatment gradually, and the value of respiratory mechanics assessment was not significantly different from control group on Day 6. In addition, CMCs were found in the lungs of mice following paraquat administration and CMCs co-treatment on Day 6, indicating CMCs may localize to the lungs and produce effects on the damaged lung milieu.
The effects of CMCs co-treatment on the damaged lung milieu were evaluated via respiratory mechanics assessment, inflammatory response and cytokine alterations. The results revealed that CMCs co-treatment significantly restored lung tissue resistance on Day 2 and Day 3 following paraquat exposure, and pulmonary compliance were recovered on Day 3. The histopathological analysis indicated CMCs co-treatment could decrease the infiltration of neutrophils into the lung parenchyma and alveolar spaces. In addition, the concentrations of cytokines associated with neutrophil recruitment and activation, including granulocyte colony stimulating factor (GCSF), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1) and keratinocyte-derived chemokine (KC), were also decreased by CMCs co-treatment.
The results of this experiment demonstrated that CMCs co-treatment could attenuate the lung damages, restore the pulmonary functions and recover the survival rate to the mice with acute fulminant paraquat poisoning, and these consequences may occur via decreasing the production of cytokines associated with neutrophils recruitment and activation and thus suppressing neutrophils infiltration to the lung parenchyma and alveolar spaces.

並列關鍵字

paraquat ； acute lung injury/acute respiratory distress syndrome ； pulmonary function ； chorion-derived mesenchymal cells ； cell therapy ； neutrophils infiltration

參考文獻

Abraham, E. (2003). Neutrophils and acute lung injury. Crit Care Med, 31: 195-199.

Addo, E., and Poon-King, T. (1986). Leucocyte suppression in treatment of 72 patients with paraquat poisoning. Lancet, 1: 1117-1120.

Afzali, S., and Gholyaf, M. (2008). The effectiveness of combined treatment with methylprednisolone and cyclophosphamide in oral paraquat poisoning. Arch Iran Med, 11: 387-391.

Ameno, K., Fuke, C., Shirakawa, Y., Ogura, S., Ameno, S., Kiriu, T., Kinoshita, H., and Ijiri, I. (1994). Different distribution of paraquat and diquat in human poisoning cases after ingestion of a combined herbicide. Arch Toxicol, 68: 134-137.

Artigas, A., Bernard, G.R., Carlet, J., Dreyfuss, D., Gattinoni, L., Hudson, L., Lamy, M., Marini, J.J., Matthay, M.A., Pinsky, M.R., Spragg, R., and Suter, P.M. (1998). The American-European Consensus Conference on ARDS, part 2: Ventilatory, pharmacologic, supportive therapy, study design strategies, and issues related to recovery and remodeling. Acute respiratory distress syndrome. Am J Respir Crit Care Med, 157: 1332-1347.