- 學位論文
熱休克效應降低砷引發人類纖維母細胞中aurora A kinase的過度表現及其後續效應
Heat shock response attenuates arsenic -induced aurora A kinase overexpression and its subsequent effects in human dermal fibroblasts
摘要
砷是一種對多重器官具高毒性與致癌性的天然元素。Aurora A kinase (aurkA)則在調節中心體分裂及成熟方面扮演重要的角色。砷的暴露之下aurk A的表現量會增加並進一步促使中心體放大，最後導致染色體不穩定以及非整倍體現象，此現象被認為與癌症的發生有高度相關。截至目前，對於砷暴露引發的生物危害並無有效的防範措施，本實驗的目的在於觀察熱休克效應在砷引起的aurk A過度表現以及其後續效應上呈現的效果。熱處理組以人類纖維母細胞於43℃下施以1小時的熱休克處理。以西方墨點法以及real time PCR分別針測aurk A的mRNA以及蛋白質表現量進行觀察。Aurk A的co-localization以及中心體放大現象則由免疫螢光染色進行觀測。實驗結果顯示，細胞暴露於0.5uM的亞砷酸鈉中72小時並不影響纖維母細胞的存活率，但會隨砷劑量的增加而遞減。以0.5 uM的亞砷酸鈉培養細胞72小時，可觀察到aurk A的蛋白質以及mRNA表現增加。若砷暴露前以熱休克處理，則細胞內過度表現的aurk A蛋白質以及mRNA則能被有效抑制。此外熱休克處理能更進一步抑制亞砷酸鈉砷所引發的中心體放大現象。根據以上結果我們推論，加熱引發的熱休克效應可藉由抑制砷暴露所引起的aurora A kinase的表現增加以及後續的中心體放大現象，而達到抑止細胞異常分裂的效果。以上結果對於防範砷暴露引起的細胞毒性提供了可能的對策。
並列摘要
Arsenic (As), a natural element which possess high toxicity and carcinogenicity. Aurora A kinase (aurk A) is a key modulator in centrosome separation and maturation states, and was found to overexpress under arsenic exposure. Overexpression of aurk A have shown to drive centrosomal amplification to further induced chromosome instability and aneuploidy, and was thought to be related to tumor induction. Until now, there is no effective way to prevent arsenic-induced biohazard. The purpose of this study was to explore the role of heat shock response on arsenic-induced aurk A overxpression and its subsequent effects. Human dermal fibroblasts of heated group were heated in 43℃ for 1 hour. We use western blotting and real-time PCR for detection of aurk A protein and mRNA level. The expression and co-localization of aurk A and level of centrosome amplification were detected by immunofluoresence. Results showed that cell viability of fibroblasts was not influenced by exposure of 0.5 uM NaAsO2 for 72 hours but decreased in a dose dependant manner while exposure to higher dosages. Exposure to NaAsO2 for 72 hours induced aurk A overexpression significantly in both protein and mRNA levels and this effect was suppressed by heat shock treatment in protein and mRNA level. Centrosome amplification caused by NaAsO2 was also inhibited by heat shock treatment. According to above result, we concluded that arsenic- induced aurk A overexpression and the subsequent centrosome amplification could be inhibited by heat shock treatment. These results highlight a potential strategy to prevent the cellular toxicity of arsenic exposure.