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    Please use this identifier to cite or link to this item: http://ir.cmu.edu.tw/ir/handle/310903500/535


    Title: 電刺激合併黃連萃取純化物小檗鹼對惡性軟骨肉瘤之腫瘤抑制作用;The Effect of Electrical Stimulation with Berberine in Chondrosarcoma Cell Line
    Authors: 許玉龍;Yu-Lung Hsu
    Contributors: 中國醫藥大學：針灸研究所碩士班
    Keywords: 電刺激;小檗鹼;惡性軟骨肉瘤;Electrical Stimulation;Berberine;Chondrosarcoma Cell Line
    Date: 2007-06-22
    Issue Date: 2009-08-11 14:24:10 (UTC+8)
    Abstract: 電刺激合併黃連萃取純化物小檗鹼對惡性軟骨肉瘤之腫瘤抑制作用
    研究生：許玉龍1
    指導教授：許昇峰1、馮逸卿2
    1.中國醫藥大學針灸研究所
    2.中國醫藥大學附設醫院骨科部
    中文摘要
    目前惡性軟骨肉瘤(chondrosarcoma)的標準治療方式仍以手術切除腫瘤為主，缺少有效的輔助化學療法，造成部份病患癒後不佳。所以研究發展出新的輔助療法對惡性軟骨肉瘤的治療是相當重要。病例報告指出合併電刺激與化療藥物可使惡性軟骨肉瘤壞死。但機轉還尚未明瞭。本實驗將合併中醫臨床常使用之電刺激與中藥萃取物小檗鹼來評估對惡性軟骨肉瘤細胞生長及凋亡的影響。
    我們使用 trypan blue exclusion assay, 4,6-diamidino-2-phenylindole dihydrochloride assays (DAPI stain)及細胞流式儀分析電刺激與中藥萃取物小檗鹼對細胞生長、細胞週期及細胞凋亡壞死之影響。小檗鹼濃度超過25μM對惡性軟骨肉瘤會依循濃度及時間而增強其毒殺細胞的效果。當以電刺激合併濃度25μM小檗鹼會使得細胞有生長抑制及細胞死亡的現象。因此電刺激具有減少小檗鹼毒殺細胞的濃度，但此作用並不會隨著電刺激的頻率增加而增加。西方墨點試驗用來檢測與細胞凋亡相關蛋白質的表現情形，如 Bax，Bcl-2，Capsase-3 及 PARP。電刺激與中藥萃取物小檗鹼會誘導細胞週期停滯於G2/M週期。並經由上升細胞內鈣離子濃度，使粒腺體過極化，因此誘導細胞凋亡相關蛋白質的表現，此機轉可能與內質網受到壓力有關。
    未來合併電刺激與中藥萃取物小檗鹼，或許，可以提供臨床上一輔助的療法，但還需要再進一步的研究。

    Chondrosarcoma are the second primary malignant bone tumor group. Surgical resection remains the primary therapy for chondrosarcoma. This mesenchymal malignancies have a poor prognosis due to the absence of an effective adjuvant therapy. Berberine has been reported to be useful in the treatment and prevention of cancer. Electrical stimulation could enhance anti-proliferation effect of chemotherapy drug. The aim of our study was to detect that electrical stimulation could enhance programmed cell death of berberine-treated chondrsarcoma cell lines and investigate the mechanism.
    The JJ cells were intervened with berberine and various frequencies of electrical stimulation, followed with incubated for 48 hours. In order to examine the effects of this intervention on cell proliferation, cell cycle, apoptosis and necrosis, we performed trypan blue exclusion assay, 4,6-diamidino-2-phenylindole dihydrochloride assays (DAPI stain)and flow cytometry analysis. Western blot was used to examine the protein levels of Bax, Bcl-2, Capsase-3 and PARP which are associated with apoptosis. Flow cytometric analysis showed that berberine with electrical stimulation significantly induced cell cycle to arrest in G2/M phase. Induction of apoptosis via ER stress involved an increase cellular Ca2+, and hyperpolarization of mitochondrial membrane and activation of associated protein before culminating in apoptosis
    In conclusion, we have demonstrated that electrical stimulation with berberine inhibits cell growth via the induction of cell cycle arrest at the G2/M phase and may induce apoptosis through the ER stress pathway in human JJ cells.
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