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研究生:郭孝美
研究生(外文):Hsiao-mei kuo
論文名稱:GST融合之endostatin對內皮和非內皮細胞上之細胞毒性
論文名稱(外文):The Cytotoxicity of GST-fused Endostatin to Endothelial and Non-endothelial Cells
指導教授:戴明泓
指導教授(外文):Ming-Hong Tai
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:80
中文關鍵詞:血管生成抑制劑cytoskeletonI血管生成endostatin膠原蛋白XVIII
外文關鍵詞:angiogenesiscollagen XVIIIangiogensis inhibitorendostatincytoskeletonI
相關次數:
  • 被引用被引用:2
  • 點閱點閱:220
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Endostatin,是一種血管生成抑制劑,在1997年由Dr. Judah Folkman’s小組發現。從他們的一系列研究中,證明是血管生成抑制作用,徹底破壞新血管的形成和導致癌細胞的餓死,是一個安全,有效抗癌方法,沒有副作用和抗藥性的發生。Endostatin在1990年進行臨床試驗階段並且在2001年完成,可以說是新一代癌症治療藥的導彈。Endostatin是細胞外膠原蛋白XVIII (collagen XVIII)經過蛋白質水解脢作用後的一個片段 (~20kDa)。它是有效抑制內皮細胞增生和血管生成,但是在其他細胞上沒有細胞毒性的影響。尤其,實驗利用遺傳工程生產出His-tagged endostatin並定期注射治療帶有腫瘤生長的老鼠,能導致腫瘤的萎縮並使腫瘤細胞處於休眠期 (dormancy),而這整個治療過程並不會導致抗藥性的發生。然而,我們對於endostatin抑制內皮細胞增生的機制仍然不清楚。利用RT-PCR從老鼠的肝臟中取得endostatin的cDNA,經過DNA定序確定後,將它殖入載體並送入E.coli中表現和產生大量的重組蛋白質GST-endostatin。GST-endostatin可溶於水,而且可以20 nM的濃度抑制內皮細胞EA.hy926的生長速率達百分之五十 (IC50),這是和His-tagged endostatin的最大不同點。在目前研究中,我們研究GST-endostatin在內皮細胞中是否會造成cytoskeleton改變。透過用一種螢光染色法再經共焦顯微鏡觀察到actin filament,發現GST-endostatin在內皮細胞中引起actin網路的破壞。西方墨點分析透露GST-endostatin以一種劑量-依賴的方法處理,產生了cytoskeleton蛋白質的減少例如tubulin、vimentin和細胞外基質相關訊息分子例如FAK、MAPK、Erk。此外,GST-endostatin減少細胞內存活因子例如AKT和NF-κB。因為GST- endostatin導致持續的鈣離子升高,GST-endostatin對protein kinase Cs (PKCs)的影響研究並顯示GST-endostatin減少PKCK1、PKC eta、PKC iota和PKC lamda。除了內皮細胞以外,研究GST-endostatin在肝癌細胞內的細胞毒性,因為collagen XVIII主要表現於肝臟,是endostatin的先驅物。想不到,GST-endostatin也能抑制肝癌細胞的增生。流式細胞儀分析和細胞核染色得知GST-endostatin能導致在肝癌細胞的凋亡小體產生。此外,GST-endostatin在肝癌細胞的差異是細胞毒性的影響,是依分化好的肝癌細胞(例如HepG2,Hep3B )和分化不好的肝癌細胞(例如Mahlavu ,Sk-hep-1),分化好的肝癌細胞IC50比分化不好的低8-10倍。尤其,GST-endostatin可以抑制Sk-Hep-1的遷移和調節Sk-Hep-1和Mahlavu細胞MMPs的分泌。總之,目前研究在GST-endostatin對細胞毒性的作用機轉中cytoskeleon網觀察到改變。此外,第一次報告了GST-endostatin對肝癌細胞增生有抑制的作用。
Endostatin, an angiogensis inhibitor, was discovered by Dr. Judah Folkman’s group in 1997. From their series studies, they demonstrated that the angiogenesis inhibition approach, which abolished the formation of new blood vessels and led to starvation of cancer cells, is a safe, effective anticancer method without side effect and drug resistance. Phase clinical trial on endostatin was carried out in 1999 and completed in 2001, heralding the approaching of a new arsenal of cancer therapy drugs. Endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on other cells. Above all, cycled therapy of experimental cancer in rodents with endostatin led to tumor dormancy without drug resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E. coli expression vector to express and generate large quantities of recombinant GST-fused endostatin. Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting endothelial cell lines EA.hy926 with a half-maximal inhibition concentration (IC50) of 20 nM. In present study, we investigated whether GST-endostatin caused alterations in cytoskeleton in endothelial cells. By using a fluorescence dye to visualize the actin filament under confocal microscope, it was found that endostatin induced the corruption of actin network in endothelial cells. Western blot analysis revealed that GST-endostatin treatment caused downregulation of cytoskeleton proteins such as tubulin, vimentin and ECM-related signaling molecules such as focal adhesion kinase (FAK), mitogen activated protein kinse (MAPK), Erk in a dose-dependent manner. Moreover, GST-endostatin decreased the levels of cell survival factor such as AKT and NF-κB. Since GST-endostatin induced sustained calcium rise, the effect of endostatin on protein kinase Cs (PKCs) were studied and revealed that endostatin reduced the levels of PKCK1、PKC eta、PKC iota and PKC lamda. Other than endothelial cell, the cytotoxicity of GST-endostatin in hepatoma cells were investigated since liver the primary expression site of collagen XVIII, precursor of endostatin. Unexpectedly, endostatin also inhibited the proliferation of hepatoma cells. Flow cytometry and nucleus staining indicated that GST-endostatin also induced apoptosis in hepatoma cells. Moreover, GST-endostatin exhibited differential cytotoxic effect against well-differentiated (such as HepG2, Hep3B) and poor differentiated (such as Mahlavu, Sk-hep-1) hepatoma cells that the IC50 for well differentiated hepatoma cells were 8-10 folds lower than for poor-differentiated cells. Above all, GST-endostatin inhibited the migration of SK-hep-1 and modulated the secretion of matrix-metalloproteinases (MMPs) by Mahlavu and SK-hep-1 cells. In summary, present study explored the role of alterations in cytoskeleon network in the cytotoxic mechanism of GST-endostatin. Moreover, the inhibitory effects of GST-endostatin on proliferation of hepatoma cells were reported for the first time.
致謝1
中文摘要3
英文摘要5
目錄7
第一章 介紹13
一. 腫瘤生物學 (tumor biology).……………..………….....13
二. 血管新生 (angiogenesis)13
三. 血管新生與腫瘤轉移14
四. 促進血管新生因子及抑制血管新生因子16
五. 癌症療法17
六. 血管新生抑制因子 (angiogenesis inhibitor)及endostatin18
七. 實驗目的和設計20
第二章 材料和實驗方法22
一. 細胞培養22
二. 蛋白質濃度分析23
三. 蛋白質電泳 (Protein electrophoresis;SDS-PAGE)..24
四. 西方墨點法 (Western Blotting)…..24
五. 細胞數目之測定25
六. 細胞增生分析…25
七. Hoechst 33258 DNA染色..26
八. Rhodamine-phalloidin染色26
九. 流式細胞儀分析細胞週期之變化27
十. Gelatin zymography..27
十一. 細胞移行分析―chemotaxis assay28
十二. 肝癌細胞collagen XVIII、NCI domain、endostatin含量測定29
第三章 結果30
一. GST-endostatin之生產30
二. GST-endostatin對內皮細胞作用.…….……………..31
1. 型態上的變化31
2. Endostatin對內皮細胞增生的影響31
3. 以螢光顯微分析GST-endostatin造成內皮細胞死亡機制32
4. 以共焦顯微分析GST-endostatin造成內皮細胞死亡機制32
三. 以western blot (西方墨點)分析endostatin造成內皮細胞死亡機制探討33
1. 蛋白質定量33
2. FAK和其磷酸化34
3. vimentin、α-tubulin和β-tubulin34
4. MAPK-P-38和Erk 1/234
5. PKC34
6. NF-kB和IkB34
7. AKT35
四. GST-endostatin對肝癌細胞作用.35
1. GST-endostatin對肝癌細胞增殖的影響.35
2. 以螢光顯微分析GST-endostatin造成肝癌細胞死亡機制36
3. 利用流式細胞儀分析GST-endostatin造成內皮細胞死亡的機制36
4. GST-endostatin對MMP-2及MMP-9釋放的影響37
5. GST-endostatinn對肝癌細胞移形的影響…..37
6. 肝癌細胞內collagen XVIII、NCI domain、endostatin含量測定38
第四章 討論39
第五章 參考文獻47
圖表53
表一:內生性調控血管生成 (angiogenesis)的物質53
圖一:血管生成 (angiogenesis)與抑制血管生成(antiangiogenesis)的過程54
圖二:Collagen XVIII、NCI domain、endostatin的結構55
圖三:GST fusion endostatin的兩種蛋白質的結構圖56
圖四:A. GST- Endostatin 蛋白質的SDS-PAGE,B.內皮細胞株EA.hy926細胞用不同濃度GST-endostatin或GST處理48小時後的型態變化57
圖五:GST-endostatin或GST對內皮細胞株EA.hy926細胞生長之劑量效應分析對數曲線圖58
圖六:內皮細胞株EA.hy926細胞經過相同濃度GST-endostatin或GST處理後DNA分裂的型態變化59
圖七:內皮細胞株EA.hy926細胞經過GST或GST-endostatin處理24小時和48小時後actin filament瓦解的型態變化60
圖八:內皮細胞株EA.hy926細胞經過GST或GST-endostatin處理後利用西方墨點分析定量蛋白質的濃度61
圖九:內皮細胞株EA.hy926細胞經過GST或GST-endostatin處理後利用西方墨點分析FAK和FAK-P的濃度62
圖十:內皮細胞株EA.hy926細胞經過GST或GST-endostatin處理後利用南方墨點分析vimentin和tubulin的濃度63
圖十一:內皮細胞株EA.hy926細胞經過GST-endostatin處理後利用西方墨點分析MAPK-P-38和Erk 1/2的濃度64
圖十二:Endostatin對內皮細胞作用經由細胞外基質的機制圖65
圖十三:內皮細胞株EA.hy926細胞經過GST-endostatin處理後,南方墨點分析PKC的濃度66
圖十四:Endostatin對內皮細胞作用經由Ca2+進入細胞內的機制圖67
圖十五:內皮細胞株EA.hy926細胞經過GST或GST-endostatin處理後利用西方墨點分析IκB和NF-κB的濃度68
圖十六:EA.hy926細胞經過GST或GST- endostatin處理後,西方墨點分析AKT的濃度69
圖十七:Endostatin對內皮細胞作用經由接受器的機制圖70
圖十八:GST-endostatin或GST對四種肝癌細胞細胞生長之劑量效應分析曲線圖71
圖十九:GST-endostatin對四種肝癌細胞細胞IC50分析結果72
圖二十:肝癌細胞株未經處理和經過GST- endostatin處理後DNA分裂的型態變化73
圖二十一:GST-endostatin對肝癌細胞之細胞週期的影響分析統計圖74
圖二十二:肝癌細胞株Sk-Hep-1和Mahlavu細胞分泌MMPs之關係研究圖75
圖二十三:肝癌細胞Sk-Hep-1細胞經過不同濃度GST-endostatin處理後的細胞移行之統計圖76
圖二十四: Hepatoma cell內collagen XVIII、NCI domain、endostatin含量測定77
表二: Heptoma cell在culture media分泌出endostatin的量79
作者簡歷80
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