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標題: 快速評估奈米材料之細胞毒性對動物細胞免疫功能與存活
Rapid evaluations of nanomaterial cytotoxicity in immune functions and survival of animal cells
作者: 鄭崇偲
Cheng, Chung-Ssu
關鍵字: titanium dioxide nanoparticles
carbon nanotubes
nano-silicate platelets
immune cells
NIH-3T3 cells

NIH-3T3 細胞
出版社: 動物科學系所
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摘要: 因奈米科技蓬勃發展,奈米材料的應用也成為現今熱門研究領域,其中奈米二氧化鈦奈米粒子(titanium dioxide nanoparticles,nano-TiO2)、奈米碳管(carbon nanotubes,CNTs)與奈米矽片(nano-silicate platelets,NSPs)已經有許多產品應用於動物生產方面。許多相關的研究證實這些奈米材料對動物健康可能存在著危害與風險,然而,卻很少研究是針對畜禽動物,尤其是對雞的研究。本試驗主要檢測 nano-TiO2(直徑約 21 nm)、CNTs(直徑為 10~30 nm,長度 2~10 μm)與 NSPs(長寬高為 80 × 80 × 1 nm3)三種奈米材料分別對雞免疫細胞中異嗜球(heterophils)與單核球/巨噬細胞(monocytes/macrophages)功能上的影響。此外,試驗中也利用老鼠胚胎纖維母細胞株(mouse embryonic fibroblast cell line)NIH-3T3 細胞作為評估奈米材料之細胞毒性(cytotoxicity)。試驗結果顯示於呼吸爆發(respiratory burst)能力上,三種奈米材料均會顯著誘導雞異嗜球釋放活性氧類物質(reactive oxygen species,ROS)(P < 0.05),但暴露於奈米材料 3 小時之單核球/巨噬細胞對於 Zymosan A 的吞噬能力(phagocytotic capacity)上則不受影響,同時高濃度的奈米材料均會顯著誘導免疫細胞分泌大量的促發炎反應細胞激素(pro-inflammatory cytokine)interleukin-1β(IL-1β)至細胞外(P < 0.05),此為發炎反應之指標。而奈米材料對 NIH-3T3 細胞之細胞毒性測試中,暴露於三種奈米材料 24 小時後,MTT(3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)分析顯示高濃度處理均會導致細胞存活率降低(P < 0.05),但在細胞凋亡路徑中 JNK(c-Jun N-terminal kinase)的活化,卻只有奈米碳管具有顯著提高其磷酸化之情形(P < 0.05)。高濃度的二氧化鈦與奈米碳管均會顯著降低細胞內抗氧化酵素 SOD(superoxide dismutase)的活性(P < 0.05),並使粒線體膜電位(mitochondrial membrane potential,MMP)顯著去極化(depolarization)(P < 0.05),而加入 ROS 清除劑(scavenger)N-MPG(n-(2-mercaptopropionyl)-glycine)也顯示其可以減緩二氧化鈦與奈米碳管所造成的粒線體膜受損(P < 0.05),但對奈米矽片處理則沒有效果。此證實二氧化鈦與奈米碳管所造成細胞毒性,氧化傷害是其中重要的一環,而奈米矽片造成細胞死亡可能不是經由 ROS 所誘發,在 annexin V 測試與電子顯微鏡鏡檢也顯示高濃度奈米矽片可能藉由包覆進而破壞細胞膜表面,最終使細胞死亡。而三種奈米材料經過測試均不會使 NIH-3T3 細胞的明顯 DNA 片段化。而飲水中添加低劑量 1000 ppb 以下的奈米矽片,不會影響雞體重變化、採食量與腸道組織形態,但劑量與處理時間還需進一步評估。綜合以上結果顯示,細胞釋放自由基與隨之的氧化傷害,可能是造成細胞毒性之原因,一旦這些奈米材料進入動物體內循環,對動物細胞存在著風險,可能影響動物健康。
Applications of nanomaterials receive more much attention recently, in which titanium dioxide nanoparticles (nano-TiO2), carbon nanotubes (CNTs) and nano-silicate platelets (NSPs) have been shown versatile functionalities in animal production. Some health-related issues have been concerned by the publics and these nanomaterials may have negative effects on animal health. However, very few studies have been tried to examine these nanomaterials effects on domestic animals, especially on chickens. We therefore investigated the effect of nano-TiO2 (diameter: 21 nm), CNTs (diameter: 10~30 nm, length: 2~10 μm), and NSPs (80 × 80 × 1 nm3) on immune responses using chicken heterophils and monocytes/macrophages as models. In addition, mouse fibroblast cell line, NIH-3T3 cells, were used to evaluate the cytotoxicity of the nanomaterials (concentration at 1, 10, and 100 μg/ml). Results showed that all of the nanomaterials induced reactive oxygen species (ROS) generation in chicken heterophils (P < 0.05) but exerted no effect on phagocytosis of monocytes/macrophages after 3 hour exposure. Moreover, there were significantly increases of pro-inflammatory cytokine interleukin-1β (IL-1β) secretion from heterophils and monocytes/macrophages (P < 0.05) when exposed to higher doses. In NIH-3T3 cells, 24 hour exposure to higher doses of nano-TiO2 and CNTs resulted in a decrease of cell viability through MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) analysis (P < 0.05), depletion of intracellular superoxide dismutase (SOD) (P < 0.05), loss of mitochondrial membrane potential (MMP) (P < 0.05). Both nano-TiO2 and NSPs nanomaterials induced cell death and this induction was not mediated by JNK/SAPK activation in NIH-3T3 cells. Treatment of n-(2-mercaptopropionyl) -glycine (N-MPG), a ROS scavenger, significantly recovered the mitochondrial depolarization (P < 0.05) in the presence of nano-TiO2 or CNTs but not in cells exposed to NSPs, suggesting that ROS generation mediates at least in part the cytotoxic mechanisms of nano-TiO2 and CNTs. Annexin V analysis and electromicroscopic imaging suggested that NSPs induced cells death by adhering and wrapping the cell surface and thereby impairing membrane integrity. The nanomaterials apparently exerted no effect on DNA fragmentation. In addition, an in vivo feeding trial for 7 days in chicken showed that NSPs failed to affect body weight, feed intake, and intestinal morphology and immune cell infiltration. Taken together, these results suggested that free radical release and following oxidative injury may account for the major cause of cytotoxicity induced by nano-TiO2 and CNTs, but NSPs exert deleterious effect by adhering to cell surface and perturbing membrane integrity. Therefore there may be potential hazards once these nanomaterials enter into the circulation of animals.
其他識別: U0005-0802201216420200
Appears in Collections:動物科學系



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