篩選新的細菌素並藉由修飾以探討其對腸癌細胞的抑制效果

Indentification and modification of two new bacteriocins from database that may have the anticancer potential.

馮政凱

指導教授 ： 林志侯

國立清華大學/生命科學院/分子醫學研究所/碩士(2013年)

摘要

近年來研究者對於抗菌胜肽(Antimicrobial peptide, AMPs)除了在抗菌能力上的發展之外，對於抑制癌細胞生長或者藥物開發上都逐漸受到重視。主要原因是抗菌胜肽來自生物體本身而非經由人類利用物理化學技術合成，故比較不容易有抗藥性的產生。細菌素(bacteriocin)則是來自細菌的一種抗菌胜肽。本篇論文從細菌篩選出可能的細菌素及其活性表現的探討。根據實驗室之前的研究指出，利用已知的細菌素結構跟特性進行分析，我們可以在NCBI、PFAM等資料庫中篩選出可能的細菌素。本實驗發現兩個基因的編碼可能為細菌素，分別是BD_21,BD_22，其存在部分Lactobacillus Casei 菌種中。
我們利用基因轉殖技術並以大腸桿菌系統生產BD21和BD22後進行活性測試。結果顯示，BD21和BD22對於實驗室現有的有害菌跟益生菌都沒有抑制效果，將BD21和BD22等比例混合後也沒發現有任何活性。因此決定針對BD21,BD22的胺基酸序列進行修飾。因為細菌及部分癌細胞之細胞膜表面的淨電荷為負電，因此將BD21和BD22的帶負電胺基酸例如:Asp、Glu利用基因技術修飾成不帶電胺基酸Ser，使淨電荷電性為正電，以此修飾增加BD21和BD22與細胞膜的結合力。結果發現修飾後的BD21m和BD22m(m表示修飾後)的抗菌能力並無顯著改變。另一方面，我們利用MTT assay測試BD21和BD22以及BD21m和BD22m對真核細胞活性的影響，結果發現不論是修飾前的BD22或修飾後的BD22m對於人類大腸癌細胞Human colon adenocarcinoma cell,SW480都有相當的毒殺效果。其中又以BD22m對SW480的毒殺效果最好，至於BD21和BD21m則沒有顯著影響。另外也發現BD22和BD22m對於Human normal fibroblast比較沒有毒殺效果。接著單染PI及雙染Annexin V、PI，並以流式細胞儀(flow cytometry)進行分析，分別可觀察細胞週期以及是否為apoptosis或necrosis。結果顯示BD22和BD22m對於SW480為apoptosis跟部分necrosis，因此未來工作將鎖定在apoptosis機制的探討。

關鍵字

細菌素 ； 抗菌胜肽 ； 大腸癌

並列摘要

Recently, scientists pay attention to Antimicrobial peptides(AMPs) due to the fact that AMPs is not only antibacteria , but anticancer and can be used in drug as well. Not produced from artificial methods, the AMPs are made by an organism. Therefore, the AMPs do not have drug resistance. The AMPs made from bacteria that is called bacteriocin. we screen a potential bacteriocin and have a test about this bacteriocin in this study. Analyzing characteristics and structures of known bacteriocins, investigated by a recent study from the lab, we screen a potential bacteriocin from database like NCBI and PFAM. In summary, we find two genes are coding about bacteriocin. The genetic codes of potential bacteriocin are BD_21 and BD_22, which exist in bacteria like Lactobacillus casei.
We produce BD21 and BD22 from E. coli by gene clonig system and test the activity . The result of activity shows that BD21 and BD22 have no activity after treated with bacteria. In addition, we do not find any activity of BD21 and BD22 after mixing BD21 and BD22 in the same proportion. Consequently, we decide to modify the peptides from amino acid sequence .Owing to the fact that bacteria and some cell membrane surface of cancer cells have negative charges, we change the amino acid which is negative of BD21 and BD22, Asp and Glu, to Ser which is no electricity, by a genetic technology. This modification can increase the interaction between BD21 and cell membrane or BD22 and cell membrane. After the modification, the antibacterial ability of BD21m and BD22m has no changes.(m means after modification)
On the other hand, we test BD21, BD22, BD21m and BD22m by MTT assay in order to know an influence of cell with these four potential bacteriocins. According to MTT assay, we find that BD22 and BD22m, can inhibit growth of the human colon adenocarcinoma cell,SW480. In particular, BD22m has the great inhibition to SW480, however, BD21 and BD21m have no influence. Furthermore, we find that BD22 and BD22m can’t inhibit growth of human normal fibroblast,HFW. Analyzing stained PI and stained Annexin V、PI by flow cytometry in order to observe cell cyle and whether the cell death is apoptosis or necrosis, we discover that BD22 and BD22m are apoptosis and some part of necrosis for SW480. Hence, a future work will focus on apoptosis mechanism and improve the activity of BD22.

並列關鍵字

bacteriocin ； Antimicrobial peptides ； human colon cancer ； flow

參考文獻

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