Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22061
標題: 志賀氏桿菌 ipaB 基因及其衍生蛋白對肺癌細胞的影響
Effects of Shigella flexneri ipaB gene and its protein product on lung cancer cells
作者: 許哲瑋
Hsu, Che-Wei
關鍵字: ipaB
肺癌
shigella
apoptosis
lung cancer
細胞凋亡
志賀氏桿菌
出版社: 分子生物學研究所
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摘要: 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。
The 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.
URI: http://hdl.handle.net/11455/22061
其他識別: U0005-2108200912393300
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