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標題: | 探討SALF、epinecidin-1及pardaxin抗菌肽之抗菌、抗發炎及抗癌活性 Study on the anti-bacterial, anti-inflammatory, and anti-cancer activities of shrimp anti-lipopolysaccharide factor (SALF), epinecidin-1, and pardaxin |
作者: | Ming-Ching Lin 林明靜 |
指導教授: | 吳金洌 |
關鍵字: | 蝦類抗脂多醣因子,石斑魚抗菌胜?,石紋豹鰨抗菌胜?,抗菌,抗發炎,抗癌, SALF,epinecidin-1,pardaxin,anti-bacteria,anti-inflammation,anti-cancer, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | 多功能抗菌胜肽具有抗菌、抗發炎及抗腫瘤的活性。在抗菌的部分,將不同片段的抗菌胜肽(蝦類抗脂多醣因子、石斑魚抗菌胜肽和石紋豹鰨抗菌胜肽)對8株細菌進行測試最小抑菌濃度值,也測試抗菌胜肽和非肽類抗生素之間的協同作用,發現抗菌胜肽(石斑魚抗菌胜肽-0217和石紋豹鰨抗菌胜肽-0127)對革蘭氏陽性和陰性菌皆具有大範圍的抗菌活性。而此兩個抗菌胜肽可以提高抗生素對於具有抗藥性金黃色葡萄球菌的作用效力,展現協同作用。
在抗發炎的部分,本研究透過酵素免疫分析法,顯示蝦類抗脂多醣因子抑制由脂多醣誘導上皮細胞產生發炎激素蛋白質,這些發炎激素包括腫瘤壞死因子(TNF)-α、白細胞介素(IL)-1α、IL-6、IL-1β,及單核細胞趨化蛋白(MCP)-1。透過反轉錄聚合酶連鎖反應,顯示蝦類抗脂多醣因子也抑制發炎激素訊息核醣核酸(il-6、il-8、il-1α及mcp-1)的產生。同時,蝦類抗脂多醣因子可能透過絲裂原活化蛋白激酶(MAPK)和轉錄因子(NF-κB)的途徑調節上皮細胞免疫反應。此外,酵素免疫分析法的結果顯示,蝦類抗脂多醣因子能降低由陰道滴蟲造成上皮細胞所分泌的發炎激素(TNF-α,IL-1α,IL-6,IL-8和MCP-1)。我們還進行反轉錄聚合酶連鎖反應的實驗,確定蝦類抗脂多醣因子影響許多發炎激素基因表達。透過西方點墨法的分析,我們確認蝦類抗脂多醣因子是透過為MAPK的路徑之一及NF-κB之路徑,抑制陰道滴蟲誘導上皮細胞的發炎反應。甚至,採用了不同的抑制劑,並且由酵素免疫分析法和西方點墨法,再次確認蝦類抗脂多醣因子的作用途徑。 另外,在抗癌的部分,研究了蝦類抗脂多醣因子對四種子宮頸癌細胞株的毒性。由流式細胞儀及細胞毒性測試結果顯示,蝦類抗脂多醣因子誘導子宮頸癌細胞死亡以及影響細胞G2/M週期。透過共軛焦顯微鏡也觀察到蝦類抗脂多醣因子在24小時處理下,會影響子宮頸癌細胞細胞質及細胞核的部分。AO/EtBr及DNA片段化實驗證實蝦類抗脂多醣因子誘導子宮頸癌細胞凋亡。此外,我們在體外用不同凋亡蛋白酶抑制劑,以及體內免疫組織染色確認蝦類抗脂多醣因子參與凋亡蛋白酶-3及-9的細胞凋亡路徑。也證明了蝦類抗脂多醣因子讓子宮頸癌細胞線粒體膜的去極化,其機制與凋亡蛋白酶的路徑有關。另一方面,也觀察到石斑魚抗菌胜肽-0217和石紋豹鰨抗菌胜肽-0127能抑制人類子宮頸癌細胞(HeLa)和纖維肉瘤細胞(HT-1080)的生長。 Anti-microbial peptides (AMPs) have many functions, such as anti-bacteria, anti-inflammation, and anti-cancer. Considering the anti-bacterial function of AMPs, we initially synthesized a series of truncated AMP derivatives of shrimp anti-lipopolysaccharide factor (SALF), epinecidin-1, and pardaxin. We then determined the minimum inhibitory concentrations (MICs) of these derivatives against eight bacterial species. We also examined the synergistic effect between peptide and non-peptide antibiotics. We found that epinecidin-0217 and pardaxin-0127 exhibited a broad range of activity against Gram-positive and Gram-negative bacteria. Moreover, a combination of these AMPs with antibiotics resulted in a synergistic improvement in the anti-bacterial activity against methicillin-resistant Staphylococcus aureus. Considering the anti-inflammatory function of AMPs, we performed ELISA to show that SALF inhibits the production of inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, and IL-1β, as well as the secretion of monocyte chemoattractant protein (MCP)-1 in cervical epithelial cells treated with lipopolysaccharide (LPS). We also conducted RT-PCR to demonstrate that SALF suppresses il-6, il-8, il-1α, and mcp-1 expressions. Moreover, we provided evidence that SALF may regulate vaginal epithelial cell immune responses via mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways. We then conducted ELISA to confirm that SALF downregulates the secretion of pro-inflammatory cytokines (TNF-α, IL-1α, IL-6, IL-8, and MCP-1) in cervical epithelial cells infected by Trichomonas vaginalis. We futher employed real-time PCR to show that SALF affects the expression of several pro-inflammatory genes. Conducting Western blot analysis, we demonstrated that SALF treatment inhibits T. vaginalis infection of cervical epithelial cells via p38 and NF-κB pathways. These pathways were observed by using appropriate inhibitors and by performing ELISA/Western blot analysis. Considering the anti-cancer function of AMPs, we investigated the cytotoxic activity of SALF on four cervical cancer cell lines. We performed fluorescence-activated cell sorting analysis to show that SALF induces cervical cancer cell death and G2/M phase arrest in vitro. Furthermore, we observed the localization of SALF to the cytosol and nuclei of human epithelial carcinoma (HeLa) cells at 24 h after treatment by confocal imaging. AO/EtBr and DNA fragmentation results indicated that SALF induces apoptosis in HeLa cells. Moreover, we used various caspase inhibitors in vitro and immunohistochemistry in vivo to show that caspase-3 and caspase-9 are involved in SALF-induced apoptosis. We also showed that SALF induces mitochondrial membrane depolarization associated with caspase-dependent pathway in HeLa cells. Our findings indicated that SALF exhibits promising anti-cancer activity by triggering apoptosis. SALF may be applied in single or combinatorial therapy against cervical cancer. We also reported that epinecidin-0217 and pardaxin-0127 inhibit the growth of HeLa and fibrosarcoma (HT-1080) cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55011 |
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顯示於系所單位: | 生化科技學系 |
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