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去氫紫堇堿及雷公藤紅素促凋亡作用研究 Study on the proapoptotic effect of dehydrocorydaline and celastrol by Xu, Zhen Tao.

English Abstract

In order to elucidate the anti-tumor effect of Corydalis yanhusuo W.T.Wang, a study on apoptotic effect of a corydalis yanhusuo W.T.Wang extract and dehydrocorydaline, a pure compound isolated from corydalis yanhusuo W.T.Wang, was carried out on human breast cancer cells -- MCF-7. In this study, methods including MTT, JC-1, Hoechst, Cell cycyle analysis, DNA Ladder and Western blot were employed. Results showed that both the extract and dehydrocorydaline could inhibit the proliferation and induce the apoptosis of MCF-7. The extract treatment would lead to cell cycle arrest at G2 phase and the significant decreased of Δψm, while the dehydrocorydaline treatment would lead to cell cycle arrest at G1 phase and no significant change of Δψm. DNA structure was destructed after treated by dehydrocorydaline for 72 h and DNA fragment could be observed in DNA Ladder. The expression of caspase-7,8 and cleaved PARP were increased and the ratio of bcl-2/bax was decreased after dehydrocorydaline treatment in MCF-7. Meanwhile, as the second part of this thesis, a study on effect of proapoptotic and DNA damage of celastrol was carried out in rheumatoid arthritis fibroblast-like synovial cells, which might play a key role in pathological development of rheumatoid arthritis. Methods including MTT, DAPI stainning, Cell cycyle analysis, Annexin-V/PI, comet assay, immuno- fluorescence and Western blot were employed. Results showed that the proliferation of fibroblast-like synovial cells was suppressd and the cell morphology was changed with the occurrence of condensed chromatin, shrunken, crimpled and condensed gray-blue nuclei after treatment. In treatment group, the percentage of annexin V-FITC binding cells significantly increased versus control group. Celastrol could induced the cell cycle arrest at G1 phase and DNA damage in fibroblast-like synovial cells. In addition, the expression of γH2AX, a marker of DNA damage, was increased. In summary, Celastrol induced the DNA V damage of FLS, which lead to G2 phase arrest, and the process of apoptosis was initiated in FLS who failed in DNA repair and retreated the cell cycle, and the proliferation of FLS was suppressed.

Chinese Abstract

爲了探索延中藥胡索抗腫瘤作用,為其臨床應用提供科學依據。本課題對延 胡索甲醇提取物及其中活性成份去氫紫堇堿對人乳腺癌細胞 MCF-7 的促凋亡作 用進行研究。實驗方法包括 MTT、Hoechst 染色、細胞週期分析、DNA Ladder、 JC-1 和 Western-blot 等。結果顯示延胡索甲醇提取物和去氫紫堇堿能夠抑制 MCF-7 細胞增殖,誘導凋亡發生。延胡索甲醇提取物誘導細胞發生 G2/M 期阻滯, 并明顯降低細胞線粒體膜電位;去氫紫堇堿誘導細胞發生 G1 期阻滯,對細胞線 粒體膜電位影響不大。去氫紫堇堿作用 72 小時后,細胞 DNA 結構被破壞,生 成斷裂的 DNA 片段,去氫紫堇堿刺激 MCF-7 細胞表達凋亡相關蛋白 caspase-7,8 及 cleaved PARP,降低 Bcl-2/BAX,而對 caspase-9 表達沒有明顯影響。 論文的第二部分為雷公藤紅素誘導人類風濕性關節炎纖維樣滑膜細胞凋亡 及 DNA 損傷的研究,旨在為雷公藤臨床治療類風濕性關節炎提供基礎理論依據。 經前期篩選,本課題以雷公藤紅素為實驗藥物,類風濕性關節炎特徵病變細胞 ——纖維樣滑膜細胞為實驗對象,採用 MTT、LDH、DAPI 染色、細胞週期分析, Annexin-V/PI 雙染、彗星電泳、免疫螢光及 western-blot 等方法,對雷公藤紅素 誘導纖維樣滑膜細胞凋亡及 DNA 損傷的作用進行研究。結果顯示,濃度為 5μM 雷公藤紅素即能明顯抑制滑膜細胞的增殖。藥物處理后,滑膜細胞形態明 顯改變,細胞核具有凋亡特徵,核偏向一極,染色質固縮,呈高亮狀態,通過 Annexin-V/PI 雙染實驗證實,細胞發生凋亡且主要為晚期凋亡。雷公藤紅素能夠 誘導滑膜細胞發生 G2/M 期阻滯,並對滑膜細胞的 DNA 造成損傷且損傷程度與 藥物濃度正相關,免疫螢光實驗顯示細胞核內標誌 DNA 損傷的特徵性蛋白γ H2AX 逐漸聚集並且含量增加。根據實驗結果,我們推測,雷公藤紅素能夠誘導 滑膜細胞 DNA 損傷,使滑膜細胞阻滯在 G2 期,G2 期 DNA 修復失敗的細胞退 出細胞週期,進入凋亡程式,最終抑制滑膜細胞的增殖。

Issue date

2013

Author

徐增濤

Faculty

Institute of Chinese Medical Sciences

Degree

M.Sc.

Subject

Cancer cells -- Growth -- Regulation

癌細胞 -- (生長 長大 發育) 之控制

Apoptosis

細胞凋亡

Breast -- Cancer

乳房 -- 癌症

Supervisor

王一濤

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1/F Zone C
Library URL
991005725309706306