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dc.contributor.authorHsu, Che-Weien_US
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dc.description.abstract痢疾桿菌Shigella flexneri 的毒性質體上帶有許多致病基因,已知其中的 ipaB 基因能夠引起巨噬細胞程序性凋亡 (apoptosis)。實驗室之前已構築一個帶有ipaB基因前803 bp片段 (ipaB803基因) 的質體,並於大腸桿菌中表現及純化出IpaB803蛋白。本研究構築另一有ipaB基因前738 bp片段 (ipaB738基因) 的質體,探討二質體所負責做出的IpaB803蛋白、IpaB738蛋白,是否對肺癌細胞有毒殺作用。 首先構築ipaB803 基因的表現載體,再將質體轉殖入四株肺癌細胞 (A549、CL1-0、CL 1-5、H460) 及一株支氣管上皮細胞株 (BEAS2B) 中,進行MTT assay以測量細胞存活率,及免疫螢光染色以觀察 IpaB803 蛋白在細胞中的表現。MTT assay的結果顯示:五株細胞的存活率皆與僅轉殖入載體的控制組相似;而免疫螢光染色的結果顯示:僅有低於 5% 的CL1-0、CL1-5細胞有IpaB803蛋白的表現;而A549、H460、BEAS2B細胞,皆沒有觀察到任何細胞有IpaB803蛋白的表現。進行免疫螢光染色實驗的同時也進行DAPI(4'',6-diamidino-2-phenylindole) 以及tubulin 的染色,發現:有IpaB803蛋白表現的CL1-0細胞,細胞tubulin的表現有減弱的情形,細胞核型態也有改變;而有IpaB803蛋白表現的CL1-5細胞,有些細胞tubulin表現正常,有些細胞tubulin則完全消失,細胞核型態則皆沒有明顯的改變。 利用大腸桿菌的表現系統,大量表現純化IpaB803蛋白與IpaB738蛋白。將純化出的IpaB803與IpaB738蛋白,以 lipofectamine包裹後,分別同樣送入四株肺癌細胞 (A549、CL1-0、CL1-5、H460) 及一株支氣管上皮細胞株 (BEAS2B)。西方雜配顯示:IpaB803、IpaB738蛋白的確有進入這些細胞株中。MTT assay結果顯示:IpaB803和IpaB738蛋白對這些細胞皆有毒殺作用,而對CL1-5的毒殺作用最強。將IpaB803蛋白以 lipofectamine包裹後,送入CL1-5細胞中,利用倒立式雷射全內反射顯微鏡,追蹤細胞型態的改變,發現有類似凋亡小泡的構造出現。 以臨床上治療肺癌之藥物 cisplatin及 etopside處理CL1-5細胞,比較不同劑量 IpaB803蛋白、IpaB738蛋白、cisplatin及 etopside毒殺細胞的效果。結果顯示:25μM etoposide可毒殺約 40% 的細胞,10μM cisplatin可毒殺約80% 細胞。 0.166μM的IpaB803蛋白即可毒殺 70%的細胞;0.178μM 的IpaB738蛋白即可毒殺 85%的細胞。zh_TW
dc.description.abstractThe virulence plasmid of Shigella flexneri contains many virulence genes and ipaB is one of them. The gene product of ipaB has been demonstrated to be capable of inducing apoptosis in macrophage. Previously, our laboratory constructed a truncated ipaB gene, called ipaB803, that encodes a protein of the first 268 amino acid residues of IpaB. In this study, another truncated ipaB gene, called ipaB738, that encodes a protein of the first 246 amino acid residues of IpaB was constructed. Effects of ipaB803 and ipaB738 and their gene products on lung cancer cells were investigated. The ipaB803 expression plasmid was also constructed and transfected into four lung cancer cell lines, A549, CL1-0, CL1-5, and H460, and one bronchial cell line, BEAS2B. MTT assay that measures viability of the cell and immunofluorescence staining that detects cells with expressed protein were performed. The results indicated that viability of the cells transfected with the ipaB803 expression plasmid was about the same as those transfected with the control plasmid. Immunofluorescence staining indicated that less than 5% of the transfected CL1-0 and CL1-5 cells expressed IpaB803 protein, and none of the transfected A549, H460, and BEAS2B cells expressed IpaB803 protein. Further DAPI and α-tubulin staining indicated that the amount of α-tubulin was low in the IpaB803-expressed CL1-0 cells with an altered nuclei morphology. While some of the expressed CL1-5 cells had a normal level of α-tubulin, others had only a very little amount of α-tubulin. The nuclei morphology of the expressed CL1-5 cells, however, was the same as the unexpressed CL1-5 cells. IpaB803 and IpaB738 proteins were over-expressed and purified in E. coli. The purified proteins were transfected into A549, CL1-0, CL1-5, H460, and BEAS2B by lipofectamine. Western analysis confirmed the existence of IpaB803 and IpaB738 in all of the transfected cells. MTT assay was then performed with the transfected cells. The results indicated that IpaB803 and IpaB738 proteins in five transfected cells reduced cell viability differently. CL1-5 cells were affected mostly. Some apoptotic body-like structures could be observed with the IpaB803-transfected CL1-5 cells under microscopic examination. Different dosages of lung cancer therapeutic drugs such as cisplatin and etopside as well as IpaB803 and IpaB738 proteins were used to treat CL1-5 cells. The results showed that 25 μM etoposide and 10 μM cisplatin reduced 40% and 80% of the cell viability, respectively. 0.166 μM IpaB803 protein reduced 70% of the cell viability, whereas 0.178 μM IpaB738 protein reduced 85% of the cell viability.en_US
dc.description.tableofcontents縮寫字對照表 i 中文摘要 ii 英文摘要 iii 前言 1 研究目的 5 材料 6 1. 細胞株與培養基 6 2. 細菌與培養基 6 3. 質體 6 4. 藥品與溶液 6 方法 6 1.細胞培養 6 2.細胞冷凍保存及解凍 6 3.細胞計數 6 4.少量細菌質體DNA抽取 7 5.DNA剪切反應 7 6.DNA純化回收 8 7.DNA黏接反應 8 8.勝任細胞製備 8 9.DNA 轉殖 8 10.DNA濃度測定 9 11.PCR 反應 9 12.15% SDS-PAGE electrophoresis 9 13.Coomassie blue stain 9 14.2D gel analysis 10 15.Silver stain 11 16.Western blot蛋白質轉印 11 17.Western blot 雜配反應 11 18.以AKTA EXPLORER 純化IpaB803、IpaB738蛋白 12 19.Protein dialysis 12 20.蛋白質定量 12 21.蛋白質轉殖 13 22.MTT assay 13 23.DNA transfection 13 24.免疫螢光染色 14 結果 15 1. 構築能表現IpaB、IpaB803、IpaB738及IpaB803- EGFP的載體 15 2. IpaB、IpaB803在四株肺癌細胞與一株支氣管上皮細胞中的表現 16 3. IpaB803-GFP在四株肺癌細胞與一株支氣管上皮細胞中的表現 16 4. 構築能在E.coli表現IpaB738蛋白的載體 17 5. 在E. coli中大量表現與純化IpaB803、IpaB738蛋白 17 6. 將純化好的IpaB803、IpaB738蛋白送入細胞內並測細胞存活率 18 7. 以IpaB803處理CL1-5細胞後,觀察細胞型態變化 18 8. 以不同濃度藥物處理癌細胞後存活率比較 19 討論 19 圖一、pFLAG-CMV2-ipaB803-His的構築 22 圖二、pFLAG-CMV2-ipaB738-His的構築 23 圖三、pFLAG-CMV2-ipaB-His的構築 24 圖四、pEGFP-N2-ipaB803的構築 25 圖五、IpaB蛋白及IpaB蛋白做突變後的蛋白的結構圖 26 圖六、以pFLAG-CMV2-ipaB803-His 轉殖至A549後細胞存活率 27 圖七、以pFLAG-CMV2-ipaB803-His轉殖至BEAS2B後細胞存活率 28 圖八、以pFLAG-CMV2-ipaB803-His轉殖至CL1-0後細胞存活率 29 圖九、以pFLAG-CMV2-ipaB803-His轉殖至CL1-5後細胞存活率 30 圖十、以pFLAG-CMV2-ipaB803-His轉殖至H460後細胞存活率 31 圖十一、以Anti-His抗體對轉殖 pFLAG-CMV2-ipaB803-His 後的細胞做Western blot 32 圖十二、以明視野或免疫螢光染色在100X視野下觀測轉殖 pFLAG-CMV2-ipaB803-His 的CL1-0細胞 33 圖十三、以明視野 或免疫螢光染色在100X視野下觀測轉殖 pFLAG-CMV2-ipaB803-His 的CL1-5細胞 34 圖十四、以免疫螢光在400X視野下觀測轉殖 pFLAG-CMV2-ipaB803-His 的CL1-0細胞 35 圖十五、以免疫螢光在1000X視野下觀測轉殖pFLAG-CMV2-ipaB803-His 的CL1-0細胞 36 圖十六、以免疫螢光在400X視野下觀測轉殖pFLAG-CMV2-ipaB803-His 的CL1-5細胞 37 圖十七、以免疫螢光在1000X視野下觀測轉殖pFLAG-CMV2-ipaB803-His 的CL1-5細胞 38 圖十八、pET-21b-ipaB803質體 39 圖十九、pET-21b-ipaB738載體的構築 40 圖二十、誘導表現IpaB803蛋白、IpaB738蛋白 41 圖二十一、破菌後IpaB803上清液通過 Ni-NTA Agarose column後, 以AKTA 及50 mM- 300 mM 梯度immidazole 液,沖出 IpaB803蛋白的沖出圖 42 圖二十二、通過Ni-NTA agarose column純化出的IpaB803蛋白, SDS-PAGE的結果圖 43 圖二十三、通 過Ni-NTA agarose column 所純化出的IpaB738蛋白, SDS-PAGE的結果圖 44 圖二十四、以Anti-ipaB803 抗體以及Anti-His 抗體對純化出的 IpaB803蛋白做western blot圖 45 圖二十五、將純化後的蛋白跑2D gel後的銀染圖 46 圖二十六、IpaB803不同分子量的2個點進行 LC/MS/MS 身分鑑定結果 47 圖二十七、將IpaB803蛋白以lipofectamine送入細胞,細胞蛋白之 western blot 48 圖二十八、IpaB803蛋白或IpaB738蛋白於96孔盤中轉殖 A549並測其存活率 49 圖二十九、IpaB803蛋白或IpaB738蛋白於96孔盤中轉殖 BEAS2B並測其存活率 50 圖三十、IpaB803蛋白或IpaB738蛋白於96孔盤中轉殖 CL1-0並測其存活率 51 圖三十一、IpaB803蛋白或IpaB738蛋白於96孔盤中轉殖 CL1-5並測其存活率 52 圖三十二、IpaB803蛋白或IpaB738蛋白於96孔盤中轉殖 H460並測其存活率 53 圖三十三、IpaB803蛋白或IpaB738蛋白轉殖A549後, 細胞型態觀察 54 圖三十四、IpaB803蛋白或IpaB738蛋白轉殖BEAS2B後, 細胞型態觀察 55 圖三十五、IpaB803蛋白或IpaB738蛋白轉殖CL1-0 後, 細胞型態觀察 56 圖三十六、IpaB803蛋白或IpaB738蛋白轉殖CL1-5 後, 細胞型態觀察 57 圖三十七、IpaB803蛋白或IpaB738蛋白轉殖H460後, 細胞型態觀察 58 圖三十八、以不同濃度etopside、cisplatin、IpaB803、IpaB738處理 A549、CL1-0、 CL1-5,48 Hr後存活率比較 59 圖三十九、利用純化出的IpaB803 ( 1μg)處理肺癌細胞株 CL1-5後細胞 型態的改變 60 圖四十、以不同濃度Etoposide處理A549、CL1-0、CL1-5細胞, 48小時後的細胞存活率 61 圖四十一、 以不同濃度cisplatin處理A549、CL1-0、CL1-5細胞, 48小時後的細胞存活率 62 圖四十二、實驗室S. flexneri菌株SH2308 的 ipaB基因序列與ATCC 發表之序列比較 63 圖四十三、實驗室S. flexneri菌株SH2308 的 ipaB基因序列與ATCC 發表之序列以及其轉譯出的胺基酸序列比較 64 參考文獻 65 附錄 69zh_TW
dc.subjectlung canceren_US
dc.title志賀氏桿菌 ipaB 基因及其衍生蛋白對肺癌細胞的影響zh_TW
dc.titleEffects of Shigella flexneri ipaB gene and its protein product on lung cancer cellsen_US
dc.typeThesis and Dissertationzh_TW
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