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    標題: 由牡丹皮所獲得之化合物於經紫外線照射之抑制黑色素生成、角質細胞之修復及抗氧化作用 由Sinularia Notanda所獲得之化合物誘導皮膚癌細胞之死亡受體凋亡途徑
    Antimelanogenesis, repair and antioxidant activities of compounds from Paeonia suffruticosa Andr. in keratinocytes exposed to ultraviolet B The death receptor-dependent apoptosis pathway of compounds from Sinularia Notanda in skin cancer cells
    作者: 陳亭宇
    貢獻者: 嘉南藥理科技大學:化妝品科技研究所
    梁家華
    關鍵字: 細胞凋亡
    軟珊瑚
    皮膚癌細胞
    抗氧化
    修復
    抑制黑色素生成
    牡丹皮
    Sinularia Notanda
    skin cancer
    Apoptosis
    Antioxidant
    Repair
    日期: 2011
    上傳時間: 2011-10-25 13:28:11 (UTC+8)
    摘要: 第一部分
      本研究由毛茛科芍藥屬之牡丹皮純化出化合物(1-6),作用於老鼠黑色素瘤細胞(B16)無明顯的細胞毒性,亦有效抑制黑色素之生成、酪胺酸酶以及多巴氧化酶之活性。化合物(1-6)藉由調節酪胺酸酶相關基因與蛋白質表現,包括黑色素皮質素受體(Melanocortin-1 receptor, MC1R)、MITF (Microphthalmia-associated transcription factor)、酪胺酸酶、TRP-2 (Tyrosinase-related protein-2)和TRP-1,進而抑制黑色素生成。此外,將人類皮膚角質株化細胞(HaCaT)、纖維母細胞(Hs68)及老鼠肝細胞(BNL CL.2)進行細胞存活度試驗(MTT)化合物(1-6)亦不具細胞毒性,並篩選出具促進細胞增生之化合物(1)、(2)、(6)進行彗星試驗,結果顯示可降低因紫外線UVB誘導HaCaT細胞所引起之細胞毒性及DNA損傷情形。化合物(1)、(2)、(6)作用於經UVB照射下之HaCaT細胞,細胞週期分佈結果顯示sub-G1期降低,S-G2/M期提升。以BrdU細胞之增殖試驗,證實化合物(1)、(2)、(6)可促進經UVB照射後HaCaT細胞增殖,化合物(1)、(2)、(6)經由調控核苷酸切除修復系統(Nucleotide excision repair, NER)之相關基因與蛋白質表現,移除經紫外線照射所引起之DNA損傷,進而促進DNA修復。將化合物(1)、(2)、(6)混合DNA後以H2O2誘導和紫外線照射,發現化合物(1)、(2)、(6)具保護DNA避免其損傷情形。進行抗氧化效能評估發現化合物(1)、(2)具清除DPPH•和ABTS•+自由基能力其具還原力效能,而在活性氧(Reactive oxygen species, ROS)試驗中,證實化合物(1)、(2)具抑制HaCaT及BNL CL.2細胞內及老鼠組織中制脂質過氧化物及活性氧(ROS)生成、促使穀胱苷肽(Glutathione, GSH)活性提升。本研究中證實由牡丹皮獲得之化合物(1-6)分別具清除活性氧和抑制黑色素,促進皮膚表皮細胞之DNA修復效能。
    第二部分
      本研究由Sinularia Notanda中純化出化合物(1-5),作用於人類上皮癌細胞(Human epithelial carcinoma cells, A431)、鱗狀癌細胞(Human head and neck squamous cell carcinoma cells, SCC25)、肺腺癌細胞(Human lung cancer cells, A549)、前列腺癌細胞(Human prostate carcinoma cells, DU145)、肝癌細胞(Human hepatocellular carcinoma cells, Hep3B),和皮膚角質株化細胞(Human skin immortalized keratinocyte, HaCaT)以及纖維母細胞(Human skin fibroblast, Hs68)反應72小時後,發現化合物(3)相較於其他化合物具明顯抑制細胞存活率,且對於A431 (IC50=1.8 M)和SCC25 (IC50 = 11.8 M)相較於對其他細胞,具有較低之作用濃度。化合物(3)對於A431和SCC25細胞之細胞毒殺作用呈現濃度依存性。經(3)作用於兩株細胞後發現細胞型態呈現凋亡(Apoptosis)典型特徵;於彗星試驗中發現DNA呈現片段化;以Annexin V/Propidium iodide染色試驗呈現細胞凋亡區域比例明顯提升;分析細胞週期變化發現週期停滯於G0/G1期,且隨著作用時間與濃度提升,sub-G1期比例增加;測定細胞中活性氧化物(Reactive oxygen species, ROS)及穀胱甘肽(Glutathione, GSH)含量,發現隨這作用時間增加,ROS含量提升,GSH含量降低。經(3)作用於兩株細胞後分析與細胞凋亡相關之基因與蛋白質調控表現,結果發現p53、Caspase-8、Caspase-9及Caspase-3表現增加;推測(3)會調控外部死亡受體凋亡路徑,促使腫瘤壞死因子(Tumor necrosis factor-α, TNF-α)和Fas ligand,結合至TNF-RI/RII (TNF-Receptor I/RII)和Fas,調節下游TRADD (TNFR-associated death domain)和FADD (Fas-associated death domain)進行細胞凋亡之訊號傳遞;(3)亦藉由活化tBid,促使內部粒線體凋亡途徑Cytochrome c和Bax表現增加,而抗凋亡因子Bcl-2表現減少。綜合上述推論(3)選擇性抑制皮膚癌細胞生長,以濃度與時間依存性誘發細胞凋亡,具有成為治療皮膚癌用藥之潛力。
    第一部分
      This study demonstrated the compound (1-6), which isolated from Paeonia suffruticosa Andr., shows non toxic in melanoma B16 cells, inhibits melanogenesis, tyrosinase activity and DOPA oxidase activity. The antimelanogenic effect of compounds (1-6)were attributable to its inhibitory abilities on tyrosinase activity, DOPAchrome formation and extracellular melanin contents, as well as down-regulated expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-2 (TRP-2) and TRP-1. Moreover, compounds (1-6) showed non toxic in human skin HaCaT cells, fibroblast Hs68 cells and mouse liver BNL CL.2 cells by MTT assay. Compounds (1), (2) and (6) promote cell proliferation, were able to attenuate the UVB-induced toxicity of HaCaT keratinocytes determined by MTT assay and comet assay.Additionally, a comparison of UVB level revealed that HaCaT cells that were incubated with compounds (1), (2) and (6) significantly reduced the sub-G1 phase and apoptosis with a concomitant arrest in the S-G2/M phase by cell-cycle distribution assay. Non-toxic compounds (1), (2) and (6) promoted HaCaT cell proliferation by increased BrdU incorporation and regulated of nucleotide excision repair (NER) in UVB-exposed keratinocytes.After treatment with UVB and H2O2 in the present of compounds (1), (2) and (6), compounds (1), (2) and (6) expressed the DNA damage protection potential from results of the electrophoretic DNA pattern. The antioxidant capacity of compounds (1) and (2) was investigated from its DPPH• and ABTS•+ free radical scavenging reactions, reducing power, and suppression of reactive oxygen species (ROS) in H2O2-induced HaCaT and BNL CL.2 cells. In this study, compounds (1-6) showed scavenging of ROS as antioxidants and suppression of melanogenesis and might promote DNA repair in the epidermal layer of the skin.
    第二部分
     The cytotoxicity demonstrated the compound (1-5) from Sinularia notanda on human epithelial cancer cells (A431), squamous carcinoma cell (SCC25), lung adenocarcinoma cell (A549), prostate cancer cell (DU145), hepatocellular carcinoma cell (Hep3B), human keratinocytes (HaCaT) and fibroblast (Hs68). The cytotoxicity results show that compound (3) expressed greater cytotoxicity to A431 (IC50 =1.8 M) and SCC25 cells(IC50 = 11.8 M) than the other compounds. Compound (3) inhibited cell proliferation, which was dose-dependent. After treatment with compound (3) in A431 and SCC25 cells, the DNA fragmentation by coment assay, the upexpression of apoptotic area by Annexin V/ Propidium iodide double stained assay, the cell cycle arrested in G0/G1 phase and increased the sub-G1phase by flow cytometry assay, increase reactive oxygen species (ROS) content and decrease glutathione (GSH) content were demonstreaed. Compound (3)-displayed apoptosis is associated with upregulation of p53, and activation of executing caspase-8, caspase-9 and caspase-3. Compound (3) revealed upregulation of tumor necrosis factor-α (TNF-α) and Fas ligand and their cognate receptors (TNF-Receptor I/II and Fas), and downstream adaptors TNF-R1-associated death domain (TRADD) and Fas-associated death domain (FADD). Moreover, compound (3) triggered activation of tBid, operation of mitochondria-mediated apoptosis pathway, including relaease of Cytochrome c and upexpression of Bax, downexpression of Bcl-2. Taken together, this study demonstrated that compound (3)-induced skin cell death was indicative of typical apoptosis and maybe potential for anti-skin cancer agent.
    關聯: 校內校外均不公開
    顯示於類別:[化妝品應用與管理系(所)] 博碩士論文

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