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題名 | 四物湯誘導癌幹細胞對化療藥物敏感性之研究=Study of Induction of Chemosensitivity in Cancer Stem Cells by Four-Agents-Decoction |
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作者 | 劉嘉燿; | 書刊名 | 中醫藥年報 |
卷期 | 3 2014.12[民103.12] |
頁次 | 頁(60)1-(60)56 |
分類號 | 414.5 |
關鍵詞 | 四物湯; 卵巢癌幹細胞; 化療抗藥性; 化療藥物敏感性; 二階段療法; Four-Agents-Decoction; Ovarian cancer stem cells; Chemoresistance; Chemosensitivity; Two-stage therapy; |
語文 | 中文(Chinese) |
中文摘要 | 抗藥性與復發在臨床上治療癌症最大的問題,癌幹細胞在癌症化學治療抗藥性扮演關鍵角色,誘導癌幹細胞對癌症化學治療敏感性有助於成功治療癌症。 目的:本計畫我們研究先給予卵巢癌幹細胞四物湯水萃物再輔以化療藥物的二階段療法,其研究之目的包含: 1.分離卵巢癌幹細胞,並證實此群細胞具癌幹細胞特性。 2.體外實驗評估四物湯與化療藥物二階段癌幹細胞治療。 3.動物實驗評估四物湯與化療藥物二階段癌幹細胞治療。 方法:1. 我們將上皮卵巢癌細胞株 SKOV-3及 OVCAR3分離具表面抗原 CD44及 CD133表現的細胞後進行增生培養,完成建立 2種卵巢癌幹細胞 SKOV3_133+44+及 OVCAR3_44+。2. 以流式細胞儀分析四物湯水萃物及化療藥物 taxol、cisplatin、doxorubicin及 lovastain對 2種卵巢癌幹細胞週期的影響,並以 MTT方法評估四物湯水萃物二階段治療或合併 (同時加入 )對其生長影響,同時分析細胞週期變化。3. 以動物體內皮下硬塊腫瘤實驗模式確認四物湯水萃物與化療藥物 cisplatin二階段療法對硬塊腫瘤的生長影響。 結果:1. 2種卵巢癌幹細胞 SKOV3 _133+44+及 OVCAR3_44+已證實具有自我更新 (selfrenewal)、抗藥性 (chemoresistance)及少細胞致腫瘤生成 (tumorgenesity)的癌幹細胞特性。2. 在二階段療法的研究,先加入四物湯水萃物的療法再佐以 cisplatin對 2種卵巢癌幹細胞可呈現藥物協同作用 (synergism),尤其在 OVCAR3_44+卵巢癌幹細胞更明顯。在第二階段中加入低濃度的 cisplatin 0.8 M即可呈現大於 70 %的抑制增生比率。四物湯水萃物與 lovastatin的二階段療法也有類似的結果,先加四物湯水萃物再佐以 lovastatin對 SKOV3_133+44+細胞具明顯藥物加成作用及劑量回應現象(dose-dependent)。但是後加四物湯水萃物的療法對 2 種卵巢癌幹細胞則會呈現藥物拮抗作用。另外,分析 2種卵巢癌幹細胞先加入四物湯水萃物的二階段療法細胞週期,發現具有 G2/M arrest的現象。在合併療法中四物湯水萃物合併化療藥物 cisplatin及 lovastatin的結果有部分不同,與 cisplatin合併對卵巢癌幹細胞呈現藥物拮抗作用 (antagonism),與 lovastatin小於 20 M濃度合併治療無明顯改變但在大於 20 M時會呈現藥物加成作用 (additivism)。3. 以動物體內皮下硬塊腫瘤實驗模式探討四物湯水萃物與化療藥物 cisplatin 二階段療法對硬塊腫瘤的生長影響。完成四物湯水萃物口服 2 g/kg 的急性毒性評估,控制組與治療組體重、血液血球、肝臟及腎臟生化指數均無明顯差異。在四物湯水萃物與化療藥物 cisplatin 對卵巢癌幹細胞腫瘤二階段療法的動物實驗,先口服給予四物湯水萃物連續 3 天後再佐以 cisplatin,共進行 4 個療程,以四物湯水萃物 0.5 g/kg 和 cisplatin 4 mg/kg 的二階段療法在第三、第四療程後可以呈現明顯的腫瘤延遲生長作用,與控制組相比分別為 66% 及 55%的抑制率,且沒有顯著的藥物副作用現象。 討論:綜合體外及體內研究成果,四物湯水萃物與化療藥物的二階段療法對卵巢癌幹細胞具有抑制生長的影響,但是其明確作用機制尚不清楚,希望未來能有機會作進一步的研究。 |
英文摘要 | Resistance to chemotherapy and recurrence is a major problem in cancer therapy. Cancer stem cells paly a major role in chemoresistance. To induce chemosensitivity in cancer stem cell paves to successful cancer chemotherapy. AIM: In this study, we use a Chinese herbal formula Four-Agents-Decoction (FAD) extract to induce chemosensitivity in ovarian cancer stem cells and then treat then with chemotherapeutic agents, so called two-stage therapy. The specific aims include: 1) isolation and identification of ovarian cancer stem cell, 2) In vitro study of two-stage therapy in ovarian cancer stem cells, 3) in animal model, in vivo study of two-stage therapy of ovarian cancer. METHODS: Using surface marker CD44 and CD133 to isolate ovarian cancer stem cells and identify the stemness of these stem cells, SKOV3 _133+44+ and OVCAR3_44+. Using flow cytometry to study the effect of FAD extract and chemotherapeutic agents on cell cycle and MTT assay to study their effect on ovarian cancer stem cell proliferation after two-stage therapy. In animal model, to study in vivo the effect of FAD extract and chemotherapeutic agents on ovarian cancer after two-stage therapy. RESULTS: Isolation and identification of two ovarian cancer stem cells, SKOV3_133+44+ and OVCAR3_44+ in terms of self renewal, chemorteistance, tumorgenicity. In vitro study of two-stage therapy, FADE and cisplatin have synergism effect in these ovarian cancer stem cells , especially in OVCAR3_44+ , more than 70% of cancer cells growth inhibition with 0.8 M cisplatin . The same effect is found in FADE and lovastatin regimen in SKOV3 _133+44+. Reversal of the two-stage therapy (chemotherapeutic agents and then FADE) results in antagonism. Cell cycle analysis reveals G2/M arrest after two-stage therapy. In combination therapy (FADE and chemotherapeutic agents given together at the same time), antagonism is found in FADE-cisplatin combination, however additivism in FADE-lovastatin combination. In mice animal model, no acute toxicity is found in animals given FADE in dose of 2 gm/kg, including animal body weight, hemogram, liver and renal function assays. In two-stage therapy, tumor is induced in these animals and then treated with FADE for three days and then chemotherapy per week cycle for four cycles. In two-stage therapy with FADE 0.5 g/kg and cisplatin 4 mg/kg, there is no side effect observed. Tumor growth inhibition of 66% and 55% was found after three and four cycles of treatment. DISCUSSION: The two-stage therapy of FADE and chemotherapy does increase the chemosensitivity in ovarian cancer stem cells in vitro and in vivo. However, the mechanism of the induction of chemosensitivity remains to be investigated. |
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