納米銀和銀離子對斑馬魚胚胎早期生長發育的影響及作用機制

為探究納米銀對水生生物的毒性作用，選取斑馬魚胚胎為受試生物，考察了納米銀對斑馬魚胚胎早期生長發育的影響，同時比較了納米銀與銀離子對斑馬魚胚胎的毒性作用和機理。實驗將受精後4小時(4 hpf)的斑馬魚胚胎分別暴露於不同濃度的納米銀和銀離子溶液中至96 hpf，觀察並記錄了胚胎的死亡、孵化和畸形等指標。應用吖啶橙(AO)染色實驗研究了胚胎暴露之後的細胞凋亡情況，並且應用螢光定量PCR技術分析了相關基因的表達水準。研究結果表明，隨著暴露濃度的增加，納米銀和銀離子均能導致斑馬魚胚胎的死亡率增加和孵化率降低，並且引起孵化延遲。納米銀和銀離子的96 h半數致死濃度(96 h-LC50)分別為11.75 mg·L⁻¹和0.054 mg·L⁻¹。銀離子毒性遠大于納米銀毒性。暴露的斑馬魚胚胎均表現出體長變短和卵黃囊腫大的畸形。AO染色結果表明，納米銀和銀離子處理組胚胎的軀幹和卵黃囊部位存在細胞凋亡信號。基因表達分析結果顯示，1.93 mg·L⁻¹納米銀顯著提高了斑馬魚胚胎caspase9的表達(P< 0.05) ，而0.006 mg·L⁻¹的銀離子就能顯著上調COX-2a(P< 0.01)和COX-17(P< 0.05)基因的表達，同時0.036 mg·L⁻¹銀離子增加了斑馬魚體內p53基因的表達(P< 0.05)。以上研究結果說明，納米銀可能通過caspase通路誘導細胞凋亡進而影響斑馬魚胚胎的生長發育；而銀離子不但影響氧化系統基因通路，還能通過p53誘導凋亡進而阻滯斑馬魚胚胎的生長發育。
To investigate the effects of silver nanoparticles(Ag NPs) on the early development of aquatic organisms and compare with that of silver ions, zebrafish embryos were selected and exposed to different concentrations of Ag NPs or Ag⁺ from 4 hours post fertilization(hpf) to 96 hpf. The survival, hatching and malformations of treated ze-brafish embryos were examined. The cell apoptosis in treated zebrafish embryos were observed by the acridine orange(AO) staining. Related genes and their expression profiles were analyzed with fluorescence quantitative PCR. Results showed that exposure to Ag NPs or Ag⁺ induced increased mortality, decreased hatching rate and delayed hatching in zebrafish embryos in a concentration-dependent manner. The 96 h half lethal concentration(96 hLC₅₀) of Ag NPs and Ag⁺ on zebrafish embryos were 11.75 mg·L⁻¹ and 0.054 mg·L⁻¹ respectively. The toxicity of Ag⁺ was greater than that of Ag NPs. Exposed zebrafish embryos exhibited shorter body length and enlarged yolk sac. The AO staining results showed cell apoptosis in the trunk and yolk sac of zebrafish were observed respectively after treatment with Ag NPs and Ag⁺. The gene expression analysis showed that Ag NPs significantly up-regulated the expression of caspase9 in zebrafish embryos at the concentration of 1.93 mg·L⁻¹ (P< 0.05). Exposure to 0.006mg·L⁻¹ of Ag⁺ significantly up-regulated the expressions of COX-2a(P< 0.01) and COX-17(P< 0.05) in zebrafish embryos. The expression level of p53 gene were also significantly increased in zebrafish embryos after exposed to 0.036 mg·L⁻¹ Ag⁺ (P< 0.05). This study suggested Ag NPs could induce apoptosis in zebrafish embryos via the caspase-dependent pathway and thus affected the development of exposed zebrafish embryos. However, exposure to Ag⁺ not only affected the oxidation-related gene pathway, but also induced apoptosis via p53 pathway and inhibited the development of exposed zebrafish embryos. Copyright © 2015 中國科學院生態環境研究中心.