- 學位論文
抗癌胜肽 KL15 藉由快速地破壞細胞膜誘導人類結腸腺癌細胞產生細胞壞死
The anticancer peptide KL15 induces necrosis and causes rapid disruption of cell membrane in a colon adenocarcinoma cell line (SW480)
摘要
近年來，抗癌胜肽的發展越來越受到重視與期待，抗癌胜肽具有篩選、誘導癌細胞死亡的能力，對於正常細胞則具有較低的副作用，而目前在臨床醫療應用仍有一些問題必須克服，例如:運送到目標細胞過程中，如何降低對周遭正常細胞的作用；如何避免被血液中所含的蛋白酶破壞。 本實驗室從Lactobacillus casei ATCC 334菌株的基因組中，篩選出具有抗菌活性的胜肽2163，實驗結果也發現其具有毒殺人類結腸腺癌細胞SW480的活性，為了更進一步的提高胜肽對細胞的毒殺能力，實驗室針對胜肽的Hydrophobic moment、Alpha-helix比例的提高，對序列進行修改，得到修改胜肽KL15。 在本篇研究中，利用CCK8 assay偵測細胞存活率，KL15對於SW480細胞的毒殺能力在16小時作用下，確實經由序列修改有明顯提升，但也發現因為序列的修改，2163與KL15對於細胞似乎產生不同的特性，實驗結果顯示，KL15在短時間內就能對SW480細胞產生毒殺作用，而原序列2163則需要更長的時間，或許是因為序列修改後誘導機制因而改變，之後我們便逐漸縮短KL15毒殺細胞作用時間進行觀察，經由共軛焦顯微鏡實驗發現，KL15在10分鐘的作用時間便能進入SW480細胞；經由CCK8 assay實驗發現，KL15在2小時內就能使SW480存活率下降36.4%，搭配Z-VAD-fmk抑制劑的使用下，細胞死亡並沒有明顯受到抑制，並且西方點墨法結果顯示在6小時的條件下，於細胞培養基中能偵測到細胞壞死的指標蛋白HMGB1，而細胞凋亡指標蛋白PARP1並沒有cleavage的現象。 本篇的研究結果顯示，KL15在非常早期就能讓SW480細胞膜受損，並進入細胞，在排除細胞凋亡的可能性後，證實細胞死亡的形式為細胞壞死。因為作用時間較短，或許在臨床使用上，能解決蛋白酶破壞的問題，在受到水解前就能發揮細胞毒殺的效果。
並列摘要
In recent years, the development of anticancer peptides attracts more and more attentions and expectations. Anticancer peptides are suggested to have the selective ability to kill cancer cells and minor side effects on normal cells. However, the use of peptides in clinical treatments is still challenging. For example, unmet needs include reducing the side effects of AMP on normal cells and preventing it from proteolysis during transport in human blood. With our efforts, we screened out the peptide 2163 with antimicrobial activity from the genome of Lactobacillus casei strain ATCC 334, and we proved that it can kill the human colon adenocarcinoma cell SW480. In order to optimize the activity to kill cancer cells, we modified the sequence of peptide 2163 of hydrophobic moments and alpha-helix ratios, resulting in the novel peptide KL15. In this study, we applied the CCK8 assay to evaluate cell viability. In 16 hours treatment, the cell-killing activity of KL15 was significantly improved because of the sequence modifications. According to our observations, the peptide 2163 and KL15 have different cell-killing characteristics. KL15 induced cell membrane disruptions in a shorter time compared with peptide 2163. Through conjugating FITC to KL15, we detected the fluorescent signals by confocal microscopy and found that KL15 can enter the cancer cell SW480 in 10 minutes. Also, the survival rate of SW480 may be reduced by 35% in 2 hours indicated by CCK8 assay, and this effect cannot be rescued by treating the caspase inhibitor Z-VAD-FMK. Furthermore, we investigated the cell-death related protein HMGB1 and PARP1 by western blot. HMGB1, the marker for cell necrosis, can be detected in cell culture media in 6 hours. PARP1, the marker for cell apoptosis, didn't show cleavage in the cells. These results show that KL15 rapidly disrupts cell membranes and induces cell necrosis. We infer that KL15 might cause cell membrane disruptions and induce cell necrosis before being proteolysis by proteases in human blood for clinical use.
參考文獻
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