- 學位論文
香菸煙霧提取物誘導細胞毒性及基因毒性在人類骨肉瘤細胞之研究探討
Cytotoxicity and Genotoxicity of Cigarette Smoke Extract on Human Osteoblastic U-2 OS Cells
摘要
抽菸早被發現為骨質疏鬆症的危險因素，但是，抽菸與骨質疏鬆症之間的確切機制仍不清楚。因此，本研究目的是以探討香菸菸霧提取物(cigarette smoke extract,CSE) 誘導人類骨肉瘤細胞 (U-2 OS) 的細胞毒性和遺傳毒性為主。本研究結果顯示， CSE處理24小時可以誘導U2-OS死亡，半致死劑量 (IC50) 為8.5%。經由彗星分析法 (comet assay) 發現CSE會誘發DNA股斷裂和DNA交叉連結，並且使用DCFDA - Cellular Reactive Oxygen Species Detection Assay發現CSE會誘導細胞內活性氧物種(Reactive Oxygen Species, ROS) 增加。另外，廣用型caspase抑制劑(BOC-D-FMK ; BOC-D(OMe)-FMK) 並無法抑制CSE誘導的細胞毒性，但N-acetylcysteine、glutathione、c-Jun N-terminal kinases抑制劑(SP600125)、Poly (ADP-ribose) polymerase 1抑制劑 (NU1025)、Necroptosis抑制劑 (Necrostatin-1) 都能有效抑制CSE誘導的細胞毒性。U2-OS細胞處理CSE經由西方墨點法分析並沒有出現Poly (ADP-ribose) polymerase 1裂解成細胞凋亡片段 (apoptosis fragments) 89kDa，但完整蛋白位置113kDa隨著CSE濃度升高而升高且出現細胞壞死片段(necrosis fragments) 55kDa。CSE誘導LC3-II蛋白質表現量增加，執行自噬作用 (autophagy)，並且能被N-acetylcysteine及glutathione的添加而抑制。經由細胞凋亡雙重染色分析技術 (Annexin-V/Propidium iodide double statining) 也顯示CSE處理讓細胞走向細胞壞死。總結，這些結果指出，CSE誘發U2-OS細胞的死亡，可能是經由reactive oxygen species產生增加，直接誘導c-Jun N-terminal kinases或造成DNA傷害誘發DNA修補酵素Poly (ADP-ribose) polymerase 1誘導receptor-interacting protein 1導致c-Jun N-terminal kinases活化，粒線體膜電位下降釋放出apoptosis inducing factor導致細胞壞死，抽菸可能是經由此途徑誘導骨質疏鬆症發生。經由DTNB分析法發現N-acetylcysteine及glutathione能夠與CSE在細胞外結合，因此N-acetylcysteine及glutathione可能可用於預防抽菸所引起的骨質疏鬆症。
並列摘要
Cigarette smoking was first identified as a risk factor for osteoporosis; however, the precise mechanism of smoking in osteoporosis remains unclear. Therefore, the purpose of this study is to investigate whether cigarette smoke extract (CSE) induces cytotoxicity and genotoxicity in Human osteosarcoma U2-OS cells. The results showed that CSE treatment for 24 hours could significantly induce the cytotoxicity and genotoxicity (DNA strand breaks and DNA cross-links) by coment assay and reactive oxygen species by DCFDA - Cellular Reactive Oxygen Species Detection Assay in U2-OS cells.In addition, the pan-caspase inhibitor BOC-D-FMK did not block CSE-induced cytotoxicity ,but N-acetylcysteine, glutathione, c-Jun N-terminal kinases inhibitor (SP600125) , Poly (ADP-ribose) polymerase 1 inhibitor (NU1025),and Necroptosis inhibitor (Necrostatin-1) can block cytotoxicity by crystal violet assay in U2-OS cells. CSE-induced increase in LC3-II protein expression performed autophagy, and can be inhibited by the addition of N-acetylcysteine and glutathione. CSE induced cell necrosis by Annexin-V/Propidium iodide double statining.The summary of these results, CSE-induced cell death of U2-OS cells through reactive oxygen species production increase, induced c-Jun N-terminal kinases directly or causing DNA damage-induced DNA repair enzyme Poly (ADP-ribose) polymerase 1 induction of receptor-interacting protein 1 lead to the activation of c-Jun N-terminal kinases, mitochondrial membrane potential release apoptosis inducing factor leading to cell necrosis, smoking may be induced osteoporosis occurs by this pathway. N-acetylcysteine and glutathione and CSE in combination in vitro by DTNB assay, N-acetylcysteine and glutathione may be used for the prevention of smoke induced osteoporosis.
並列關鍵字
cigarette smoke extract ； reactive oxygen species ； U-2 OS ； PARP-1 ； DNA damage ； JNK ； autophagy ； necrosis參考文獻
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