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研究生:洪淑敏
研究生(外文):Shu-Mine Hung
論文名稱:靈芝促進人類肝癌細胞凋亡之研究
論文名稱(外文):Studies on the apoptotic effects of Ganoderma species on human hepatoma cells
指導教授:孫璐西孫璐西引用關係蕭明熙蕭明熙引用關係
指導教授(外文):Lucy Sun Hwang, Ph. D.Ming-Shi Shiao, Ph. D.
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:129
中文關鍵詞:細胞凋亡靈芝
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靈芝促進人類肝癌細胞凋亡之研究
洪淑敏
國立台灣大學食品科技研究所
摘 要:
肝癌為近年來國人十大死因之首,缺乏早期診斷及有效藥物治療乃為高死亡率之主要原因。甲羥戊酸為所有甲羥戊酸多重途徑中異戊二烯代謝物的共同前驅物,其對細胞正常生長代謝功能的維持是重要且多樣的。研究顯示,甲羥戊酸生成之抑制可降低膽固醇生合成及抑制Ras、G-protein異戊二烯化,若甲羥戊酸更嚴重降低,則細胞週期停止、DNA複製受抑制且誘導細胞凋亡。肝癌細胞的生長代謝遠較一般正常細胞快,且需仰賴甲羥戊酸多重途徑生成較多之膽固醇維持生長,若甲羥戊酸多重途徑受到抑制,則癌細胞的生存亦受抑制。本研究以對肝有高度特異性的降膽固醇藥物HMG-CoA reductase競爭性抑制劑-Lovastatin來抑制甲羥戊酸多重途徑,使細胞處於趨向細胞凋亡狀態之G0/G1期,在此狀態下,若加入促進細胞凋亡之藥物(如Taxol),則細胞將更易走向細胞凋亡。本研究採用缺乏p53與Bcl-2,但可表現MDR-1基因的人類肝癌細胞珠Hep 3B細胞為離體模式,探討當甲羥戊酸的量已降低至膽固醇生成受抑制且細胞生長停止之階段下,添加篩選自靈芝Methanol抽出物的細胞凋亡促進劑,是否可增加Hep 3B細胞對該細胞凋亡促進劑之敏感度,並以裸鼠接種肝癌細胞Hep 3B/T2為動物腫瘤模式,驗證其活體抗腫瘤藥理效果。
結果顯示,依抑制Hep 3B細胞生長之活性為依據,靈芝Methanol抽出物GL-M1-0820及其各階段之層析區分物GL-M1-0820-M1及GL-M1-C-M-14分別對Hep 3B細胞抑制其存活率,其IC50值分別為10.1 g/mL、34.3 g/mL及13.5 g/mL,而Hep 3B細胞先經Lovastatin 10 M處理48小時後,再處理GL-M1-0820、GL-M1-0820-M1及GL-M1-C-M-14 72小時,則造成Hep 3B細胞凋亡,其IC50值分別為2.2 g/mL、4.0 g/mL及7.1 g/mL,顯示Lovastatin配合靈芝區分物前後處理之效果更顯著於靈芝區分物單獨處理。另採流式細胞分析儀探討細胞週期,顯示上述活性區分物均使Hep 3B細胞生長停止於G2/M期,且在處理72小時後造成細胞凋亡,依誘導細胞凋亡生成計畫性死亡小體的活性(48小時)由高至低其序為GL-M1-C-1H2E-5-RH-5>GL-M1-C-M-14-C-9>GL-M1-C-M-14>GL-M1-0820。動物實驗顯示,對照組(Ct)、Lovastatin處理組(L)、GL-M1-C-M-14單獨處理組(C14)、先用Lovastatin後用GL-M1-C-M-14協同處理組(LC14)之腫瘤大小分別為3.041.61 cm3、2.242.38 cm3、1.281.03 cm3及0.910.71 cm3,經統計分析結果顯示Ct組與C14組(p=0.048)且Ct組與LC14組均達顯著性差異(p=0.014)。
本研究可獲得以下結論:Hep 3B細胞在其甲羥戊酸途徑受抑制的狀態之下,可更有效地被誘導細胞凋亡。靈芝天然物中具有可抑制Hep 3B細胞於G2/M期且造成細胞凋亡的有效成份,值得進一步探討應用於肝癌輔助藥物治療的可行性。
Studies on the Apoptotic Effects of Ganoderma Species
on Human Hepatoma Cells.
Shu-Mine Hung, Graduate Institute of Food Science
and Technology, National Taiwan University
Abstract
Hepatocellular carcinoma (HCC) is the second cause of cancer mortality in Taiwan recently. The high mortality of HCC is due to poor early diagnosis and lack of effective therapy. Mevalonate is the obligatory precursor of isoprenoids in the multiple mevalonate-demanding pathways and is vital for diverse cell function. Previous studies have shown that the inhibition of mevalonate formation decreases cholesterol biosynthesis and inhibits protein isoprenylation including Ras and G-protein. Further reduction of mevalonate pool causes cell cycle arrest, inhibition of DNA replication, and cell apoptosis. As a working hypothesis of this study, we speculate that the demand of mevalonate to support hepatoma cells proliferation are higher than that for normal hepatocyte regeneration. The subsistence of hepatoma cells in the liver will be inhibited preferentially when hepatic pool of mevalonate for multiple mevalonate-demanding pathways are reduced. The study intends to inhibit the multiple mevalonate-demanding pathways by using the very liver specific, cholesterol-lowering drug lovastatin, a HMG-CoA reductase inhibitor. With the inhibition of the multiple mevalonate-demanding pathways by lovastatin, the cells will be arrested at the G0/G1 phase, a cell cycle stage closer to apoptosis. In this situation, the hepatoma cells are expected to be more sensitive to other apoptotic agents. Human hepatoma cell line Hep 3B was chosen as the in vitro model. Hep 3B cells are p53 and bcl-2 deficient, but can express the MDR-1 gene. Previous studies from this laboratory and others have strongly indicated that the Chinese medicinal fungus Ganoderma species, which are commonly used as adjuvant therapy in HCC patients, was chosen to elucidate the apoptotic effect. Bioassay guided active fraction with significant apoptotic effect in vitro was used for animal study to elucidate the pharmacological potential. In vivo study used Hep 3B/T2 hepatoma cell-implanted nude mice as the animal model.
Results showed that GL-M1-0820, GL-M1-0820-M1, GL-M1-C-M-14, and GL-M1-C-1H2E-5-RH-5 were the fractions from Ganoderma spp. which reduced the viabilities of Hep 3B cells. Their IC50 values were 10.1 g/mL, 34.3 g/mL, 13.5 g/mL, and 7.3 g/mL, respectively. If pre-treated cells with 10 M lovastatin for 48 hr, Hep 3B cells became more sensitive to GL-M1-0820, GL-M1-0820-M1, and GL-M1-C-M-14 (subsequent treatment for for 72 hr). The IC50 values were 2.2 g/mL, 4.0 g/mL, and 7.1 g/mL, respectively. The results indicated that the combination treatment with lovastatin and Ganoderma fractions was more effective than those treated with Ganoderma fractions alone. Cell cycle determination by flow cytometry indicated that these active fractions arrested Hep 3B cells at G2/M. Animal study demonstrated that the tumor sizes of control (Ct, n=6), lovastatin (L, n=6), GL-M1-C-M-14 (C14, n=6), and sequential treatment of lovastatin and GL-M1-C-M-14 (LC14, n=6) groups were 3.04 ± 1.61 cm3, 2.24 ± 2.38 cm3, 1.28 ± 1.03 cm3 and 0.91 ± 0.71 cm3, respectively. The reduction of tumor size by combination treatment in a sequential manner was most effectively.
In conclusion, growth-arrested human hepatoma cells (Hep 3B) became more sensitive to apoptotic agents if the mevalonate pool was reduced. The Chinese medicinal fungus Ganoderma contained apoptotic natural products, which are most likely oxygenated triterpenes, promoted hepatoma cell apoptosis in vitro and in vivo. This study may shed light on better chemotherapy to human HCC and potential application of Ganoderma as the adjuvant therapy for HCC.
目 錄
頁數
壹、 前言………………………………………………………….... 1
貳、文獻整理………………………………………………………. 3
一、肝癌………………………………………………………. 3
二、細胞凋亡…………………………………………………. 7
三、膽固醇生合成途徑………………………………………. 17
四、靈芝………………………………………………………. 23
參、研究設計………………………………………………………. 32
肆、材料與方法……………………………………………………. 35
一、材料儀器與實驗動物……………………………………. 35
(一) 靈芝藥材……………………………………………. 35
(二) 藥品與耗材…………………………………………. 35
(三) 實驗細胞……………………………………………. 37
(四) 實驗動物……………………………………………. 37
(五) 儀器設備……………………………………………. 37
二、靈芝抽出物之製備………………………………………. 38
(一) 溶劑萃取……………………………………………. 38
(二) 矽膠管柱分離GL-M1-0820……………………….. 38
1. 以矽膠吸附GL-M1-0820………………………. 38
2. 配製移動相(Mobile phase)………………….. 39
3. 矽膠管柱充填…………………………………… 39
4. 矽膠管柱層析(Silica gel column chromatography)
………………………………………………………. 40
5. 分配萃取………………………………………… 40
(三) 矽膠管柱分離GL-M1-C-M-14…………………… 40
1. 配製移動相…………………………………….. 40
2. 矽膠管柱充填………………………………….. 40
3. 矽膠管柱層析(Silica gel column chromatography)
…………………………………………………….. 41
三、靈芝Methanol區分物高效液相層析法………………. 41
四、靈芝活性成分之分析與鑑定………………………….. 42
(一) 質譜儀………………………………………….. 42
(二) 氫核磁共振(1H-NMR)……………………… 43
五、溶液之配製……………………………………………... 43
(一) Phosphate-buffered saline………………………. 43
(二) TEG solution……………………………………. 43
(三) Hank’s balanced salt solution…………………… 44
(四) Dulbeccos Modified Eagles Medium………….. 44
六、人類肝癌細胞株Hep 3B細胞及Hep G2細胞之培養與保存
(一) 培養條件……………………………………….. 44
(二) 保存…………………………………………….. 45
七、靈芝成份及試劑對於細胞生長之影響……………….. 45
(一) MTT 呈色分析法……………………………… 45
(二) 細胞密度與MTT呈色關係之標準曲線製備… 46
(三) 酒精為助溶劑對Hep 3B細胞之影響………… 46
(四) 靈芝成份對細胞生長及形態之影響………….. 47
(五) 協同Lovastatin及靈芝成份之處理…………… 47
(六) Lovastatin及靈芝成份抑制Hep G2細胞合成膽固醇之能力………………………………………….. 48
(七) 靈芝成份對細胞週期(Cell cycle)所造成之影響
…………………………………………………… 48
八、推算動物實驗Lovastatin及GL-M1-C-M-14之使用劑量 49
(一) Lovastatin………………………………………… 49
(二) GL-M1-C-M-14………………………………….. 50
九、動物實驗…………………………………………………. 50
(一) 第一次動物實驗…………………………………. 50
(二) 第二次動物實驗…………………………………. 51
十、統計分析…………………………………………………. 52
伍、結果………………………………………………………….. 54
一、 靈芝Methanol抽出物…………………………………. 54
二、 GL-M1-0820對於人類肝癌細胞株Hep 3B細胞所造成之影響……………………………………………………….. 54
(一)細胞生長及型態……………………………….. 54
(二)細胞合成膽固醇能力………………………….. 54
(三)細胞週期……………………………………….. 55
三、 GL-M1-0820經管柱層析所得區分物對Hep 3B細胞之影響
(一)管柱層析分離GL-M1-0820…………………... 55
(二)酒精為助溶劑對Hep 3B細胞之影響………... 55
(三)GL-M1-0820經管柱層析所得區分物對細胞之生長與型態之影響…………………………………………….. 56
(四)GL-M1-0820之管柱層析區分物對Hep G2細胞合成膽固醇能力之影響…………………………………….. 58
(五)GL-M1-0820之管柱層析區分物對Hep 3B細胞之細胞週期之影響………………………………………….. 58
四、 GL-M1-0820協同Lovastatin處理對於人類肝癌細胞株Hep 3B細胞所造成之影響…………………………………. 59
五、 動物實驗……………………………………………… 60
(一) 第一次動物實驗……………………………… 60
(二) 第二次動物實驗……………………………… 61
陸、討論……………………………………………….. 62
一、 離體模式之合理性……………………………………. 62
二、 協同處理之探討………………………………………. 63
三、 動物實驗結果探討……………………………………. 63
四、 結論……………………………………………………. 64
柒、參考文獻…………………………………………………… 65
圖 次
頁數
Fig. 2-1. Estimates of the HBsAg seroprevalence in the world’s populations……………………………………………….. 6
Fig. 2-2. Modulation of apoptosis in therapy of human liver diseases. 9
Fig. 2-3. Signals that activate p53…………………………………… 12
Fig. 2-4. Transcriptional targets of p53, and their interactions with apoptosis signalling proteins……………………………… 13
Fig. 2-5. The p53-Mdm2 autoregulatory loop………………………. 15
Fig. 2-6. Mitochondrial apostat: regulation of the Bcl-2 family of apoptosis regulators………………………………………………….. 16
Fig. 2-7. Mevalonate pathway………………………………………. 19
Fig. 2-8. HMG-CoA reductase inhibitors (Statins) used as cholesterol-lowing drugs……………………………………………… 20
Fig. 2-9. Cytotoxic ganoderic acids reported by Toth et al………… 30
Fig. 2-10. Cytotxic lanostanoids and steroids of Ganoderma tsugae. 31
Fig. 5-1. Calibration of HepG2 and Hep 3B cell number by the MTT method……………………………………………………. 102
Fig. 5-2. Effects of methanolic extract of Ganoderma spp. on the growth of Hep 3B cells…………………………………………… 103
Fig. 5-3. RP-HPLC profiles of GL-M1-C-1H2E-5…………………. 104
Fig. 5-4. The thin layer chromatographic profiles of GL-M1-C-1H2E-5 and GL-M1-C-1H2E-5-RH-5. …………………………… 105
Fig. 5-5. RP-HPLC profiles of GL-M1-C-1H2E-5-RH-5. …………. 106
Fig. 5-6. Effects of the de-lipidated methanolic extract of Ganoderma spp. on the growth of Hep 3B cells. …………………………… 107
Fig. 5-7. Effects of GL-M1-C-M-14 separated from GL-M1-0820 by silica gel column chromatography on the growth of Hep 3B cells……………………………………………………… 108
Fig. 5-8. RP-HPLC profiles of GL-M1-C-M-14-C-1E1M-9……… 109
Fig.5-9. Effect of the methanolic extract of Ganoderma spp. on the growth of Hep 3B cells after 48 hr lovastation treatment.
…………………………………………………………… 110
Fig. 5-10. Effect of the de-lipidated methanolic extract of Ganoderma spp. on the growth Hep 3B cells after 48 hr lovastation treatment.
…………………………………………………………… 111
Fig. 5-11. Effects of GL-M1-C-M-14 obtained from GL-M1-0820 by silica gel column chromatography on the growth of Hep 3B cells after 48 hr lovastatin treatment. ……………………. 112
Fig. 5-12. Effect of taxol and various fractions from Ganoderma spp. and incubation time intervals on morphological changes of Hep 3B cells (×400 under inverted stage microscope equipped with phase contrast)…………………………………………… 113
Fig. 5-13. Effects of taxol and GL-M1-0820 on the cell cycle distribution of Hep 3B cells. …………………………………………. 117
Fig. 5-14. Effects of GL-M1-C-M-14, GL-M1-C-M-14-C-1E1M-9 and GL-M1-C-1H2E-5-RH-5various treatments on the cell cycle distribution of Hep 3B cells……………………………… 118
Fig. 5-15. Effects of taxol and various fractions from Ganoderma spp. on the cell cycle distribution of Hep 3B cells……………….. 119
Fig. 5-16. The percentage of sub G1 population in Hep 3B cells treated with taxol and various fractions from Ganoderma spp. with time……………………………………………………….. 122
Fig. 5-17. Tumor growth in nude mice treated with lovastatin and GL-M1-C-M-14 alone and in combination…………………… 123
Fig. 5-18. Tumor growth in nude mice treated with lovastatin and GL-M1-C-M-14 alone and in combination. ………………….. 125
Fig. 5-19. Tumor growth in nude mice treated with lovastatin and GL-M1-C-M-14 alone and in combination…………………… 126
Fig. 5-20. Tumor growth in nude mice treated with lovastatin and GL-M1-C-M-14 alone and in combination…………………… 127
表 次
頁數
Table 2-1. Risk factors of liver cancer and estimates of the attributable fractions………………………………………………… 4
Table 2-2. 靈芝之生理功效……………………………………….. 29
Table 5-1. Effect of methanolic extract of Ganoderma spp. on the growth of Hep 3B cells……………………………………….…. 77
Table 5-2. Separation of the methanolic extract of Ganoderma spp. by silica gel column chromatography……………………… 78
Table 5-3. Effect of lovastatin on the growth of Hep 3B cells. …….. 79
Table 5-4. Effects of combined treatment of taxol and ethanol on the growth of Hep 3B cells after 48hr lovastatin treatment. 80
Table 5-5. Effects of column fractions of GL-M1-0820 on the growth of Hep 3B cells. …………………………………………… 81
Table 5-6. The inhibition activities of Hep 3B cell viability by column fractions separated from GL-M1-0820 by using silica gel column chromatography. ……………………………….. 82
Table 5-7. Effects of fractions of GL-M1-C-1H2E-5 by RP-HPLC on the growth of Hep 3B cells. ………………………………… 83
Table 5-8. Effects of the de-lipidated methanolic extract of Ganoderma spp. on the growth of Hep 3B cells. ……………………. 84
Table 5-9. Effect of fraction GL-M1-C-M-14 on the growth of Hep 3B cells. …………………………………………………….. 85
Table 5-10. The inhibition activity of Hep 3B cell viability by GL-M1-C-M-14. ……………………………………………………. 86
Table 5-11. Separation of GL-M1-C-M-14 of the methanolic extracts of Ganoderma spp. by silica gel column chromatography…. 87
Table 5-12. Effects of various column fractions of GL-M1-C-M-14 on the growth of Hep 3B cells. ………………………………… 88
Table 5-13. The inhibition activities of Hep 3B cell viability by column fractions obtained from GL-M1-C-M-14 by using silica gel column chromatography. ……………………………….. 90
Table 5-14. Effects of fractions separated from GL-M1-C-M-14-C-1E1M -9 by RP-HPLC on the growth of Hep 3B cells…………. 92
Table 5-15. Effects of the methanolic extract of Ganoderma spp. on the growth of Hep 3B cells after 48 hr lovastatin treatment… 93
Table 5-16. Effects of the de-lipidated methanolic extract of Ganoderma spp. on the growth of Hep 3B cells after 48 hr lovastatin treatment. ……………………………………………….. 94
Table 5-17. Effects of GL-M1-C-M-14 separated from GL-M1-0820 by silica gel column chromatography on the growth of Hep 3B cells after 48 hr lovastatin treatment. …………………… 95
Table 5-18. Effects of lovastatin and GL-M1-0820 to inhibit cholesterol biosynthesis in Hep G2 cells as determined by incorporation of (2, 3H) acetate into (3H) cholesterol. …………………. 96
Table 5-19. Effects of column fractions of GL-M1-0820 to inhibit cholesterol biosynthesis in Hep G2 cells as determined by incorporation of (2, 3H) acetate into (3H) cholesterol. ….. 97
Table 5-20. Tumor growth in nude mice treated with lovastatin and GL-M1-C-M-14 alone and in combination. ………………… 98
Table 5-21. Tumor growth in nude mice treated with lovastatin and GL-M1-C-M-14 alone and in combination………………….. 100
附件:本研究所採用之Ganoderma spp.…..……………………… 129
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