- 學位論文
抗菌胜肽BD21在SW480細胞株啟動細胞凋亡及壞死程序
Apoptotic and necrotic process in SW480 cells triggered by an antimicrobial peptide BD21
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摘要
近年來，抗菌胜肽的研究除了抑菌之外，抗癌的潛力越來越受到重視與投入。本實驗室從Lactobacillus (L.) paracasei ATCC 27092菌株的基因序列篩選出帶有GXXXG motif的Class II抗菌胜肽BD21，利用PCR技術我們將此序列插入表現載體pAB中。MTT實驗發現，BD21具有誘發腸道癌細胞SW480進行細胞凋亡潛力。除此之外，流式細胞儀結果顯示BD21也會誘發腸道癌細胞SW480進行壞死。本次實驗利用西方墨點法測定各種與細胞凋亡相關的蛋白表現量，預測可能的凋亡路徑。實驗結果顯示，BD21處理人類腸癌細胞SW480後，誘發細胞的caspase-8、caspase-7、caspase-9、caspase-3活化， Bid會造成粒線體釋出AIF、cytochrome C、Smac等凋亡相關蛋白，證實細胞凋亡也會走向粒線體介導途徑。除此之外，SW480 necrosis marker cyclophilin A蛋白表現量也明顯上升。綜合以上的結果，推論BD21可能會直接或間接啟動相關受器導致細胞凋亡路徑的蛋白活化，造成細胞凋亡及壞死。
並列摘要
We searched the genomic database (NCBI) and found a gene which encodes a Class IIb bacteriocin plus with GXXXG motif in Lactobacillus (L.) paracasei ATCC 27092, named BD21. We amplified this DNA fragment by PCR and ligated with an overexpression vector pAB to give pAB-BD21. The MTT assay result showed that BD21 can inhibit the growth of human colon adenocarcinoma cell, SW480. Moreover, flow cytometry analysis also showed that BD21 cause apoptosis and partly necrosis in SW480. Although we found that BD21 can induce apoptosis and partly necrosis in SW480, the mechanisms are not clear. In this study, we show that the levels of caspase-8, caspase-7, caspase-9, caspase-3 and SW480 necrosis marker cyclophilin A had significantly increased. Moreover, Bid may contributed to released the AIF, cytochrome C and Smac from the mitochondria to the cytosol according to the results of Western blot and the SW480 necrosis marker cyclophilin A are also increased. In summary, BD21 may indirectly or directly activate the death receptor and cause apoptosis and necrosis in SW480 cell.
參考文獻
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