利用斑馬魚進行奈米毒性評估及利用CRISPR-Cas9 工具編輯斑馬魚抑癌基因

Evaluation of nanotoxicity in zebrafish and CRISPR-Cas9 mediated genome editing of tumor suppressor genes in zebrafish

史云佳(Sreeja Sarasamma)

指導教授 ： 蕭崇德

中原大學/理學院/化學研究所/博士(2019年)

https://doi.org/10.6840/cycu201900616

摘要

中文摘要

碳富勒烯納米顆粒（NP）是天然存在的並且廣泛用於各種製造工業中。雖然先前的研究已證明富勒烯暴露後具有肺和皮膚細胞的毒性，但碳納米顆粒在神經行為中的潛在傷害作用大部分尚未被探究。在這裡，我們評估了C60和C70 NPs暴露對成年斑馬魚的行為，神經病理學和生化反應變化的影響。兩個不同的暴露劑量用於該實驗：低劑量（0.5ppm）和高劑量（1.5ppm）。在富勒烯NPs暴露兩週後進行行為測試。我們發現在C60和C70 NPs暴露2週後的魚中，兩個時間點的運動，探索和群游活動減少以及焦慮和晝夜節律受損。神經病變如病理變化和神經炎症也存在於C70 NPs暴露的魚的大腦中。生化分析的結果顯示以下觀點：在斑馬魚暴露於1.5ppm的C60和C70 NPs後，腦和肌肉組織中活性氧、超氧化物歧化酶、皮質醇和Hif1α的活性顯著增加。此外炎症標誌物如ssDNA，TNF-α和IL-1β的含量也顯著增加。經C70處理的魚的生化變化也可藉著組織學觀察到。最後，我們使用行為表型組學方法，特別注重於對環境的影響，來評估金屬和碳NPs的毒性，此法也可以很容易地轉變應用於其他水生魚類。總之這是首次的研究，利用成年斑馬魚幾種神經行為參數的變化來說明暴露於環境相關量的富勒烯後的毒性。
根據最近對台灣臨床資料的調查，在抑癌基因中，tp53（40-85％），brca2（40-80％），brip1（10％）和chek2（20％）基因變異被報導。由於我們可以找到與轉移相關的有害突變，但這些癌症突變在癌症發展中的真正作用尚不清楚。為了監測致癌作用的每個步驟，我們嘗試在斑馬魚中開發基因敲除模型。在致癌誘導後，斑馬魚可以自發性地發展腫瘤。為了研究抑癌基因在腫瘤發生中的作用，我們應用CRISPR / Cas9技術編輯這些選定的基因而建立了5個具有移碼突變(frame-shift mutation)的斑馬魚品系。

關鍵字

富勒烯 ； 納米毒性 ； 表型組學 ； CRISPR / Cas9(常間回文重複序列叢集關聯蛋白) ； 斑馬魚

並列摘要

ABSTRACT

The articles included in this thesis cover five studies. The first and second study was focused on the evaluation of Carbon fullerene toxicity in adult zebrafish. Carbon fullerene nanoparticles (NPs) are naturally occurring and widely utilized in the various manufacturing industry. While previous studies have demonstrated toxicity in lung and skin cells after fullerene exposure, the potential detrimental role of carbon NPs in neurobehavior is largely unaddressed. Here, we evaluated the effects of C60 and C70 NPs exposure on behavior, neuropathology, and alterations in biochemical responses in adult zebrafish. Two different exposure doses were used for this experiment: low dose (0.5ppm) and high dose (1.5ppm). Behavioral tests were performed after two-week exposure of fullerene NPs. We found decreased locomotion, exploration and shoaling activities at both time points as well as anxiety and circadian rhythm impairment after 2 weeks in both C60 and C70 NPs exposed fish. The results of biochemical assays indicate the following notion: following the exposure of zebrafish to 1.5ppm of C60 and C70 NPs, the activities of reactive oxygen species, superoxide dismutase, cortisol and Hif1 in brain and muscles tissues increased significantly. Finally, we used an approach of behavioral phenomics with a particular focus on environmental influences, which can also be easily adapted for other aquatic fish species, to assess the toxicity of metal- and carbon-based NPs. The third study was focused on the microplastic toxicity in zebrafish. The fourth chapter reveals the low concentration of zinc chloride inhibits brain acetylcholine level and produces neurotoxic signatures in zebrafish. The final study was an investigation of the role of tumor suppressor genes in cancer development in zebrafish. According to the recent survey of Taiwan clinical data, among the tumor suppressor genes, tp53(40-85%), brca2 (40-80%), brip1(10%) and chek2(20%) were reported. To investigate the role of tumor suppressor genes in tumorigenesis, we established five zebrafish lines with a frameshift mutation of these selected genes by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)/ associated protein 9) technology.

並列關鍵字

fullerenes ； nanotoxicity ； phenomics ； CRISPR/Cas9 ； zebrafish

參考文獻

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