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研究生:陳嘉琪
論文名稱:順雙氨雙氯鉑、輻射線、及亞砷酸鈉在非小細胞肺癌H1299細胞的細胞毒性機制研究
論文名稱(外文):The cytotoxic effects of cisplatin, x-ray, and sodium arsenite in non-small cell lung canaer-H1299 cells
指導教授:黃海美
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:57
中文關鍵詞:順雙氨雙氯鉑亞砷酸鈉非小細胞肺癌
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在近年報導中指出,以transient方式轉殖正常p53基因可以提高細胞對藥物的反應。 本研究則採用轉殖正常p53方式選出穩定表現p53的細胞,再作細胞對藥物反應的研究。
將建構有正常p53與neo基因的質體轉殖入無p53蛋白表現的null-p53 H1299的細胞,挑選到能持續表現p53蛋白的兩株細胞 (S16以及S40)。 其中有較多量p53調控的下游基因p21以及Mdm2蛋白質表現的S40細胞,選定作為對應null-p53 H1299細胞,作為後續細胞毒性研究。
分別處理cisplatin、X-ray、或亞砷酸鈉 (SA) 再分別以: (A) SRB (sulforhodamine B) 染色細胞方式與 (B) 流式細胞技術比較p53-H1299-S40細胞及null-p53 H1299細胞的細胞毒性結果。 在SRB細胞毒性結果中,以50%抑制生長濃度值 (IC50) 為基準,在cisplatin處理組,轉殖p53的H1299細胞與null-p53 H1299細胞之IC50各為0.8μM和1.8μM,在敏感程度上,前者為後者的2.3倍;在X-ray處理組,ID50分別為2.2Gy與3.8Gy,有轉殖p53的細胞與無轉殖者敏感程度比約為1.7倍;在SA處理組,IC50分別為5.5μM與12μM,有p53轉殖的H1299細胞敏感程度較無轉殖的細胞株高2.2倍。
使用Hoechst33258與PI螢光染色細胞樣品,再以流式細胞技術分析結果獲得:有轉殖p53的H1299細胞在分別處理上述的三種試劑後,所得凋亡細胞的百分比都明顯增加。 處理4μM cisplatin組有22%;照射X-ray 6Gy組有28%;而處理20μM亞砷酸鈉組有27%。
分別處理上述三種試劑後,檢查參與細胞程序性死亡相關的蛋白質p53、Bak、caspase3表現方面,結果如下:處理任何一種試劑,轉殖p53的H1299細胞之p53表現都有隨著處理劑量提高而增加;在低濃度處理下有明顯的caspase 3的表現。 相同的處理方式下,對應null-p53 H1299細胞則完全沒有caspase 3表現。 至於,Bak蛋白質表現方面,三種處理組,所得結果各有不同:在處理X-ray或亞砷酸鈉組中,轉殖p53的細胞,Bak的表現量皆隨著提高劑量而明顯增加。 至於cisplatin處理組,在有無轉殖p53的H1299細胞中,Bak的表現並無不同。
本研究成功轉殖正常p53於原來無p53表現的null-p53 H1299細胞中並建立一支含有穩定p53表現的H1299-S40細胞株,以SRB細胞毒性與流式細胞技術測度,此細胞對cisplatin、X-ray、與SA的敏感度以及細胞凋亡程度都比null-p53 H1299高。 與細胞程序性死亡有關的蛋白分子p53、Bak (除了處理cisplatin組)、caspase 3也都有顯著的表現。 因此,轉殖p53的H1299細胞株,在分別處理cisplatin、X-ray、及亞砷酸鈉後,所造成的敏感度提高,可能是透過活化p53調控之細胞程序性死亡路徑所致。 此外,cisplatin處理組造成的細胞凋亡可能與Bak調控的路徑無關。 未來,進一步可使用本實驗所建立的細胞株模式,可提供在測試其他藥物及試劑的反應或p53分子機制相關的基礎研究上。
To determine whether p53 plays a role as a modulator of chemo- and radio- therapeutic agents in NSCLC, the stable transfection of wild-type p53 into H1299 cells were performed. In this study, the role of both wild-type and null p53 in modulating the sensitivity of H1299 cells to three therapeutic agents, cisplatin, X-ray, and sodium arsenite, is examined.
The sensitivity to the three agents was tested in the sulforhodamine B (SRB) cell viability assay. The data showed that H1299 cells transfected with wild-type p53 gene had an increase in susceptibility to the three agents (2.3-fold to cisplatin, 1.7-fold to X-ray, and 2.2-fold to sodium arsenite) in comparison with parental or neo-transfected H1299 cells.
Furthermore, the apoptotic cell fraction was also determined by dual-parameter flow cytometry after treatment with the three agents. By growing cells in the presence of cisplatin, X-ray, and sodium arsenite, respectively, the apoptotic fractions of H1299-S40 cells increased to 22%, 28%, and 27%, respectively.
The expressions of p53, Bak (pro-apoptotic protein) and caspase3 (apoptotic proteolytic enzyme) were examined by Western blot. In p53-transfected H1299-S40 cells p53 proteins were obviously increased after treated individually with the three agents. The results showed similar Bak protein expression was found in both p53-transfected H1299-S40 and null-H1299 cells following cisplatin treatment, but not sodium arsenite treatment or X-ray exposure. In comparison, the active patterns of caspase 3 protein in H1299-S40 cells after above mentioned three agents were dramatically expressed but not in parental H1299 cells. These indicated that the p53 enhanced sensitivities to above three agents in cells through Bak-dependent or Bak-independent (cisplatin) apoptotic pathway.
These observations suggest that the increase in chemo- or radio-sensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In the future, the model cell line system could be used in other drugs investigation or p53 molecule related research.
中文摘要 1
Abstract 3
Introduction 5
The purpose of this study 14
Materials and methods 15
Results 23
Discussion 30
References 35
Figures 39
Appendix 1 56
Appendix 2 57
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