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標題: 志賀氏桿菌ipaB基因產物對哺乳動物細胞毒殺作用之探討
The cytotoxic effects of Shigella flexneri ipaB on mammalian cells
作者: 陳琬蓉
Chen, Wan-Rong
關鍵字: Shigella flexneri;志賀氏桿菌;ipaB gene;mitochondria;ipaB基因;粒線體
出版社: 分子生物學研究所
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Shigella flexneri為革蘭氏陰性細菌,具有毒性質體,其上帶有許多包括ipaB致病基因。本實驗室有pFLAG-CMV2-IpaB-His和pFLAG-CMV2-IpaB803-His質體,分別可在哺乳動物細胞表現重組 Flag-IpaB-His 蛋白及缺C端hydrophobic domain的Flag-IpaB803-His蛋白。本研究首先將此二質體轉殖到HepG2、CL1-5、CHO-K1三株細胞株中,培養48小時後,以免疫螢光染色觀察重組蛋白的表現。結果顯示: 將pFLAG-CMV2-IpaB-His質體送入HepG2、CL1-5、CHO-K1,表現FLAG-IpaB-His蛋白的細胞平均百分比為<1%、<2%、~10%;而將pFLAG-CMV2-IpaB803-His質體送入此三株細胞株,表現FLAG-IpaB803-His蛋白的細胞平均百分比為<1%、10%、15%。
pFLAG-CMV2-IpaB-His質體送到HepG2、CL1-5、CHO-K1、A549、HeLa,培養24小時後,除了HeLa細胞存活率增加26%,其他細胞存活率皆不變 (與送pFLAG-CMV2質體到三株細胞的對照組相比);培養48小時後,五株細胞的存活率為減少15%、不變、不變、增加20%、不變。送pFLAG-CMV2-IpaB803-His質體到五株細胞,培養24小時後,細胞存活率皆不變;培養48小時後,五株細胞存活率分別為減少15%、減少20%、不變、不變、不變。
送pFLAG-CMV2-IpaB-His 到CL1-5,24、48、72小時後,進行免疫螢光染色。發現送入pFLAG-CMV2-IpaB-His質體,24、48、72小時後,有FLAG-IpaB-His蛋白表現的CL1-5細胞,細胞型態改變。24小時後,有FLAG-IpaB-His蛋白表現的細胞,一半FLAG-IpaB-His蛋白在細胞質,一半FLAG-IpaB-His蛋白在細胞質/細胞核;48小時後,全部細胞FLAG-IpaB-His蛋白在細胞質;72小時後,全部細胞FLAG-IpaB-His蛋白在細胞質/細胞核。另一方面,24、48小時後,約二成的細胞,其細胞核有濃縮的現象,八成細胞,細胞核正常;72小時後,細胞核皆正常,但細胞膜有小泡的生成 (blebbing)。
送pFLAG-CMV2-IpaB-His和pFLAG-CMV2-IpaB803-His到CL1-5,48小時後,利用MitoTracker dye染色,觀察細胞粒線體的型狀。發現有FLAG-IpaB-His蛋白表現的細胞,粒線體型狀不變;但是有FLAG-IpaB803-His蛋白表現的細胞,粒線體明顯片斷化。
將可在細胞表現IpaB-EGFP及IpaB803-EGFP蛋白的pEGFP-N2-IpaB及pEGFP-N2-IpaB803質體轉殖到HEK-293T、CL1-0、PA-1、CHO-K1、CL1-5,48小時後,有表現IpaB-EGFP及IpaB803-EGFP蛋白的CHO-K1細胞平均百分比為<5%,有表現IpaB803-EGFP蛋白的HEK293T細胞平均百分比為<5%,表現二蛋白的其他四株細胞百分比為<1%;但送入pEGFP-N2質體到五株細胞,表現EGFP蛋白的五株細胞百分比,皆為30% ~ 40%。顯示IpaB-EGFP蛋白及IpaB803-EGFP蛋白在五株細胞株中可能易被降解,導致有蛋白表現的細胞百分比低 (<1% 或5%)。

Shigella flexeri is a gram negative bacterium, which contains the virulence plasmid including IpaB gene. pFLAG-CMV2-IpaB-His and pFLAG-CMV2-IpaB803-His plasmids were transfected into mammalian cells, which can expression FLAG-IpaB-His recombinant protein and absence of C-terminal hydrophobic domain of FLAG-IpaB803-His protein. In this study, the HepG2, CL1-5, CHO-K1 cell lines containing the two plasmids were cultured to 48 hours and using immunofluorescence staining, recombinant protein expression was studied. The results showed that the FLAG-IpaB-His protein in the three cell lines showed <1%, 5%, 10% protein expression whereas the FLAG-IpaB803-His protein showed <1%, 10%, 20%.
HepG2, CL1-5, CHO-K1, A549, HeLa cell lines contanining pFALG-CMV2-IpaB-His plasmid was cultured for 24 hrs showed 26% increased survival rate in HeLa cells but no difference in the other cell lines. For the 48 hrs, HepG2 showed 15% reduced survival rate but A549 showed 20% increased survival rate. pFLAG-CMV2-IpaB803-His plasmid was transferred to the 5 cell lines and cultured for 24 hrs, showed no difference in the cell survival while 48hrs showed 15% and 20% reduced survival rate in HepG2 and CL1-5 respectively.
The two plasmids were transferred to CL1-0, U937, CaCO-2 and COLO205 cell lines and cultured for 24 hrs and 48 hrs. Cell survival was the no difference.
The pFLAG-CMV2-IpaB-His plasmid was tranfected in CL1-5 cell line and protein expression was studied using immunofluorescence staining. The CL1-5 was cultured for 24 hr, 48 hr, and 72 hr. After 24 hrs, half of the FLAG-IpaB-His protein was found in the cytoplasm and other half in the cytoplasm/nucleus; 48 hrs, FLAG-IpaB-His protein in all the cells was found in the cytoplasm; 72 hrs, FLAG-IpaB-His protein was found in the cytoplasm/nucleus in all cells. It was found out that FLAG-IpaB-His protein expression inhibited the cell differentiation of the CL1-5. On the other hand, after 24 hr and 48 hr, 20% of the cells show nuclei condensation. After 72 hrs, the nucleus is normal but showed blebbing in the cell membrane.
pFLAG-CMV2-IpaB803-His plasmid was transferred to CL1-5 cell line and cultured to 24 hr, 48 hr, and 72 hrs. After 24 hrs, half of the FLAG-IpaB-His protein is found in the cytoplasm and the rest in the cytoplasm/nucleus. In 48 hrs, 70% of the protein is in cytoplasm and 30% in the cytoplasm/nucleus. In 72 hrs, all the protein is in the cytoplasm. Apoptotic bodies can be seen in the 48 hrs and 72 hrs. Cell differentiation is inhibited by the expression of FLAG-IpaB803-His protein. On the other hand, after 24 hrs, nucleus is condensed in 50% of cells but after 48 hr and 72 hrs, half of the cell's nuclei are condensed and the rest have broken nucleus.
The CL1-5 line was transfected with pFlAG-CMV2-IpaB-His and pFLAG-CMV2-IpaB803-His to check the effect of the protein expression to the mitochondria. It was found that the FLAG-IpaB-His protein did not change the shape of the mitochondria but the FLAG-IpaB803-His protein caused mitochondrial fragmentation.
The pEGFP-N2-IpaB and pEGFP-N2-IpaB803 plasmids were transfected into HEK-293T, CL1-0, PA-1, CHO-K1, CL1-5 cell lines and cultured for 48 hrs. The expression of IpaB-EGFP and IpaB803-EGFP recombinant protein expression was studied. The percentage of the 2 proteins in CHO-K1 cells is <5% but in the other cell lines is <1% but the pEGFP-N2 plasmid to the 5 cell lines contains 30% to 40% of EGFP protein. This shows that expression of IpaB-EGFP protein and IpaB803-EGFP protein in the five cell lines is susceptible to degradation resulting in the low levels of protein expression.
CL1-5 cell line containing the pEGFP-N2-IpaB and pEGFP-N2-IpaB803 plasmid was cultured for 24 hr and 48 hr. The cell survival was the no different but the recombinant protein expression inhibited the cell differentiation but showed no fragmentation of the mitochondria.
其他識別: U0005-2208201114304300
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