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  • 學位論文

抗癌中藥材之研究 第七報 天然單寧成分之抗子宮頸癌活性

Antitumor Activity of Chinese Drugs VII The Antitumor Activity of Natural Tannins on Cervical Carcinoma

指導教授 : 顏焜熒 楊玲玲
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摘要


子宮頸癌位居國內女性生殖器官癌症死亡原因之第一位,臨床治療除以手術、放射線療法外,亦有以化學藥物來治療,而臨床上使用之抗癌藥物常有抗藥性及副作用的問題,故新抗癌藥物之開發在癌症治療上乃 刻不容緩之課題。 本實驗以子宮頸癌細胞株(HeLa cell line)為標的,應用trypan blue及MTT細胞染色法,建立子宮頸癌藥物體外篩選模式,並進行天然物之篩選。結果子宮頸癌細胞株(HeLa cell line)在96 well plate之最適生長濃度為40000 cells/ml,其兩倍生長時間為57.02小時。在加藥後第三天為較佳觀察時間。 生藥為藥物開發的來源之一。本實驗選擇由本實驗室所抽取、純化之在37種tannins天然物來作抗子宮頸癌的體外活性篩選,結果以hirtellin B處理48小時之IC50為14.7 mM,抑制活性較明顯,且其對癌細胞的選擇性較高。因此由檉柳中純化、分離hirtellin B,來進行體外細胞毒性機轉之探討。 體外細胞毒性機轉試驗以分子生物學和細胞型態學兩方面來進行探討。前者利用放射線物質[3H]thymidine、[3H]uridine、[3H]leucine)嵌入HeLa 細胞,探討其對DNA、RNA和蛋白質生合成之影響。結果顯示,hirtellin B是因造成細胞毒性而導致細胞死亡,進而導致核酸、核糖核酸及蛋白質之合成減少,且具有濃度依存性。另外,利用流式細胞儀(flow cytometry)分析hirtellin B對細胞週期的影響,結果發現並無明顯之抑制。 在細胞型態學研究方面,利用光學顯微鏡及電子顯微鏡觀察HeLa 細胞之型態變化,結果在光學顯微鏡下以Giemsa-White染色法,發現細胞質有空泡的產生,但細胞核無明顯之變化;而以電子顯微鏡觀察其超顯微構造,發現細胞質有空泡的產生,粒線體有膨大的現象,但細胞核無明顯之變化。 綜合以上之結果,推論hirtellin B對HeLa細胞具有細胞毒性,且屬細胞周期非特異性藥物。

並列摘要


Carcinoma of the cervix is one of the most common malignancies in women worldwide. In clinical, cervical cancer is curable in most patients by surgery, radiotherapy, or chemotherapy. However, we found many drugs used for chemotherapy caused resistance and have many side effects in clinical. Therefore, it is urgat to develop new antitumor drugs. In this study, we used the human cervical carcinoma cell line (HeLa cell line) to establish a system for cytotoxic compounds screening by using trypan blue staining and MTT. We found that the most suitable cell concentration which cells in 96 well plate is 40000 cells/ml. The doubling time of HeLa cells is 57.02 hours. The test time for observation is three days after treated with drug. Chinese medicine and natural products are the sources to develop new antitumor drugs. In this paper 37 kinds of natural tannins were surveyed the cytotoxic activity to cervical canner cell line-HeLa. The results of cytotoxic effects of the tested tannins, hirtellin B (HB) exhibited the most cyotoxic potency to HeLa cell line. The IC50 of HB was 14.7 mM by treated for 48 hours. And it's selective index is more than the others. Therefore, we isolated and purifiied hirtellin B for the study of its mechanism. The DNA, RNA, and protein syntheses were measured by the cellular incorporation of 3H-thymidine, -uridine, -leucine. We found that hirtellin B lead HeLa cell death by it's cytotoxicity, and leaded the DNA, RNA, and protein biosyntheses decreased. And the distribution of DNA content in the cell population was evaluated by flowcytometry, and found that it has no significant inhibition to the cell cycle of HeLa clls. Light-microscopic and electron- microscopic (TEM)studies were applied to find that it has vacular on cytoplasma, and mitochondria swelling. But no significant change on cell nucleus. The above results showed that hirtellin B had cytotoxic effect to HeLa cell, and it is a non-specifical cell cycle drug.

參考文獻


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