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研究生:曹予馨
研究生(外文):Tsao, Yu-Hsin
論文名稱:紫蘇葉萃取物的治療潛力之研究
論文名稱(外文):Study on Therapeutic Potential of Perilla Leaf Extract
指導教授:李家惠王翊青
指導教授(外文):Lee, Chia-HueiWang, I-Ching
口試委員:洪錦堂邱慶豐
口試委員(外文):Horng, Jim-TongChiu, Ching-Feng
口試日期:2023-12-29
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2023
畢業學年度:112
語文別:中文
論文頁數:35
中文關鍵詞:紫蘇葉口腔鱗狀細胞癌咖啡酸迷迭香酸介白素-1β表皮生長因子接受器艾瑞莎順鉑
外文關鍵詞:perillaoral squamous cell carcinoma (OSCC)caffeic acidrosmarinic acidinterleukin (IL)-1βepidermal growth factor receptor (EGFR)gefitinibcisplatin
相關次數:
  • 被引用被引用:0
  • 點閱點閱:16
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  • 下載下載:3
  • 收藏至我的研究室書目清單書目收藏:0
紫蘇葉萃取物已被證實具有多種藥理特性。研究結果顯示紫蘇葉萃取物能抑制口腔鱗狀細胞癌(以下簡稱為口腔癌)的細胞生長,且其生長抑制力正比於口腔癌細胞之惡性。經由高效液相層析分析法得知咖啡酸和迷迭香酸是紫蘇葉萃取物中主要的酚類成分,對口腔癌細胞TW2.6的72小時之半抑制濃度分別是362.3 μM和173.7 μM;對於OC3之半抑制濃度分別為477 μM和280.8 μM。咖啡酸和迷迭香酸混合能夠劑量依賴性地抑制口腔細胞株OC3與TW2.6的生長。當兩者總濃度介於300 μM‒500 μM區間時,咖啡酸和迷迭香酸以1:2比例混合有最佳抑制力,然而兩者並無協同作用。酵素連結免疫吸附分析法結果顯示咖啡酸、迷迭香酸或兩者混合均能有效抑制OC3細胞原發及腫瘤壞死因子-α激發後介白素-1β的生成。西方墨點法分析結果顯示OC3細胞生長於含有咖啡酸、迷迭香酸或兩者並存的培養液中,會增強OC3細胞本身及受腫瘤壞死因子-α激發後表皮生長因子接受器的活化,同時提高OC3細胞對低劑量 (5 μM) 表皮生長因子接受器酪氨酸激酶抑製劑艾瑞莎的敏感度。此外,集落形成實驗結果顯示口腔癌細胞株長期培養於含有迷迭香酸、咖啡酸與迷迭香酸混合物的培養液中可有效提高低劑量(1 μM)順鉑對其生長的抑制力。此外,流式細胞儀分析結果顯示口腔癌細胞OC3以咖啡酸(100 μM)、迷迭香酸(200 μM)或兩者混合處理72小時可誘導細胞凋亡。
綜合以上實驗結果,我們認為紫蘇葉中的咖啡酸和迷迭香酸可以作為口腔癌艾瑞莎標靶治療及順鉑化療的輔劑,有效提高口腔癌細胞對艾瑞莎和順鉑的敏感度。此研究成果不僅是紫蘇葉酚類成分藥效的新發現,也具有臨床應用潛力。
The herbal medicine perilla leaf extract (PLE) has various pharmacological properties. Our study showed that PLE inhibits the proliferation of oral squamous cell carcinoma (OSCC) cells and the proliferation inhibitory effect correlates with the malignancy of OSCC. High performance liquid chromatography analysis revealed that caffeic acid (CA) and rosmarinic acid (RA) constitutes the two main components of perilla phenols. After treatment of 72 h, CA and RA inhibits the viability of OSCC cells in a dose-dependent manner. The IC50 value for CA and RA in inhibiting the viability of TW2.6 cells were 362.3 μM and 173.7 μM, respectively. The IC50 value for CA and RA in inhibiting the viability of OC3 cells were 477 μM and 280.8 μM, respectively. The viability of TW2.6 and OC3 cells also suppressed by the mixture of CA and RA. The optimal CA/RA combination ratio was 1:2 at concentrations of 300–500 μM but had no synergistic effect on inhibiting the viability of OSCC cells. Results from enzyme-linked immunosorbent assay showed that CA, RA, or CA/RA mixture significantly suppressed OC3 cells to production of interleukin-1β (IL-1β) in the presence or absence of Tumor Necrosis Factor-α (TNF-α) stimulation. Western blot analysis indicated that treatment of CA or the combination of CA and RA enhanced the activation of EGFR in OC3 cells in the presence or absence of TNF-α stimulation. Moreover, treatment with CA or the combination of CA and RA also increased the sensitivity of OC3 cells to low-dose (5 μM) EGFR tyrosine kinase inhibitor gefitinib. Colony forming experiments showed that RA or the combination of CA and RA enhanced the growth-inhibitory effect of low-dose (1 μM) cisplatin. Furthermore, flow cytometry analysis indicated that CA (100 μM), RA (200 μM), or the combination of CA and RA induced apoptosis of OC3 cells after 72 h‒incubation.
In summary, these results suggest that the perilla phenols, CA and RA, can serve as adjuvants in gefitinib targeted therapy and cisplatin chemotherapy of OSCC, to enhance the sensitivity of OSCC cells to gefitinib and cisplatin. Findings from this study not only revealed a novel pharmaceutical effect of perilla phenols but also uncovered the potential of perilla phenols for clinical application.
中文摘要-ii
Abstract-iii
Contents-iv
Chapter 1. Introduction-1
Chapter 2. Materials and Methods-4
1.Cell culture-4
2.Preparation of PLC-4
3.Cell viability assay, thiazolyl blue tetrazolium bromide (MTT)-5
4.High performance liquid chromatography (HPLC) analysis of PLE-5
5.Western blot analysis-6
6.Determination of IL-1β levels in culture media-7
7.Colony forming assay-7
8.Apoptosis assay-8
9.Quantitative real-time PCR (qPCR)-8
Chapter 3. Results-10
1.PLE selectively suppressed viability of OSCC cells-10
2.Identification of phenols in PLE-11
3.Antiproliferation effects of CA and RA on OSCC cells-11
4.CA and RA alone or in combination suppressed the release of IL-1β from TNF-α-induced or non-induced OC3 cells-12
5.CA and RA alone or in combination induced EGFR activation and enhanced antitumor activity of low-dose gefitinib-13
6.Chronic CA and RA treatment enhanced the cytotoxicity of low-dose cisplatin in TW2.6 cells-15
7.CA and RA alone or in combination induced apoptosis in OC3 cells in a time-dependent manner-16
Chapter 4. Discussion-17
Chapter 5. Conclusion-23
Reference-24
Figures-27
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