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研究生:陳妙佩
研究生(外文):Miao Pei Chen
論文名稱:血管增生抑制因子vasostatin引發內皮細胞凋亡並抑制老鼠腫瘤生長
論文名稱(外文):Vasostatin induced apoptosis in endothelial cells and inhibited Lewis lung carcinoma in mice
指導教授:陳世杰陳世杰引用關係戴明泓
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:81
中文關鍵詞:血管新生血管新生抑制因
相關次數:
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摘 要
近幾年來,抗血管新生的策略已經積極地被應用研究於癌症的治療,一種最新被鑑定出來的血管新生抑制因子vasostatin,從EBV-immortalized 細胞株的條件培養液分離出,並鑑定為calreticulin (與鈣離子結合的蛋白質) 之N端。在這個研究當中,我們首先利用RT-PCR的方法,從淋巴瘤細胞中選殖vasostatin cDNA,經由DNA定序比對鑑定後,再將vasostatin cDNA送入含有六個組氨酸的載體,且經由大腸桿菌來表現及產生大量重組vasostatin,經由實驗數據顯示vasostatin為可溶性,並可以抑制牛動脈之內皮細胞的增殖,它只要以2 nM的濃度即可以抑制BAEC (牛動脈之內皮細胞)的生長速率達百分之五十,但在 3T3 (纖維母細胞)為無效的。在流式細胞儀及利用Hoechst33258染色之螢光顯微分析,顯示vasostatin引發內皮細胞的凋亡,除此之外,於動物體內新生血管實驗結果顯示,注射vasostatin (200 mg/egg)可抑制雞胚絨毛膜之新生血管,及注射vasostatin (100 mg/mice)於帶有Lewis-lung carcinoma老鼠,不但可抑制帶有Lewis-lung carcinoma老鼠之腫瘤生長,此外在相同的劑量下,vasostatin於抑制內皮細胞增殖及老鼠的腫瘤尺寸中明顯的比endostatin增強許多並有統計上的差異,這個發現似乎和先前的研究是一致的,先前報告指出vasostatin有效的劑量為endostatin及 angiostatin的4-10倍,因此這種抗血管新生的分子對於人類癌症的預防及治療可能是有益的 。
Abstract
Anti-angiogenesis strategy has been actively investigated for cancer treatment in recent years. One the latest identified angiogenesis inhibitors, vasostatin, was isolated from conditioned media of EBV-immortalized cells and identified as NH2-terminal fragment (amino acids 1-180) of a calcium-binding protein calreticulin. In this study, we have cloned vasostatin cDNA from lymphoma cells by RT-PCR. After verification by DNA sequencing, vasostatin cDNA was subcloned into E.coli expression vector to express and generate large quantities of recombinant 6xHis-tagged vasostatin. The 6xHis-tagged is soluble and capable of inhibiting proliferation of bovine aortic endothelial cells (BAEC) with IC50~ 40 nM without significant adverse effect on 3T3 cells. Flow cytometry and fluorescence microscope analysis by Hoechst 33258 indicated that vasostatin induced apoptosis in BAE cells. Besides, injection of vasostatin (200 mg/egg) inhibited in vivo angiogenesis in chicken chorioallantoic membrane (CAM) and suppressed tumor growth in mice bearing Lewis lung carcinoma cells. Moreover, potency of vasostatin in inhibition of neovascularization in CAM and tumor size in mice is significantly stronger than that of endostatin at the same dose. This finding seems to be consistent with previous study which reported the effective dose of vasostatin is 4-10 fold lower than that of endostatin and angiostatin. Therefore, this potent anti-angiogenesis molecule may be useful for the prevention and treatment of human cancers.
目 錄
摘 要1
Abstract3
誌 謝4
目 錄5
第1章 前言9
1-1 血管新生 (angiogenesis)9
1-2 腫瘤生物學 (Tumor biology)9
1-3 生長因子及生長抑制因子10
1-4 血管新生與腫瘤轉移11
1-5 癌症療法12
1-6 血管新生抑制因子 (angiogenesis inhibitor)及vasostatin12
1-7 化學治療 (chemotherapy)及血管增生抑制劑 (angiogenesis inhibitor)14
1-8實驗目的15
第2章 材料與方法16
2-1 B淋巴球核酸之萃取16
2-2 於大腸桿菌中表現並純化重組的vasostatin20
2-3 鑑定蛋白質純度及大小22
2-4 細胞培養27
2-5 蛋白質於細胞實驗分析29
2-6 體內 ( in vivo )試驗36
第3章 結果39
3-1 反轉錄聚合酵素連鎖反應檢測 ( RT-PCR )39
3-2 重組蛋白 (recombinant vasostatin)的表現與純化39
3-4 測試vasostatin對內皮細胞形態上的變化40
3-5 測試vasostatin對內皮細胞增殖的影響40
3-6 測試vasostatin抑制內皮細胞增殖的時間點41
3-7 測試vasostatin對纖維母細胞形態上及增殖的影響41
3-8 利用CAM來評估新血管生成(neovascularization)41
3-9 以Lewis lung carcinoma於BL57/6 mice做tumor angiogenesis的實驗42
3-10利用流式細胞儀分析vasostatin造成內皮細胞死亡的機制42
3-11以螢光顯微分析vasostatin造成內皮細胞死亡的機制43
3-12 vasostatin對內皮細胞移形的影響43
3-13 vasostatin對MMP-2及MMP-9釋放的影響44
第4章 討論45
4-1抗血管藥物與其他療法之比較45
4-2 vasostatin相關文獻46
4-3 calreticulin在細胞所扮演的角色46
4-4 結合療法47
4-5重組蛋白質的純化及功能分析48
4-6 機制的探討與未來展望50
第5章 參考文獻52
圖表56
表1.內生性調控血管生成及抗血管生成因子。56
圖1. 血管生成或新生血管的過程。57
圖2. vasostatin位於calreticulin之N端包括1-180個氨基酸。58
圖3. DNA電泳分析vasostatin之RT-PCR產物。59
圖4. vasostatin cDNA之DNA序列。60
圖5. SDS-PAGE分析重組 vasostatin之純度與大小。61
圖6. 內皮細胞株BAEC細胞經過不同濃度的 vasostatin或PBS處理24小時後的形態變化。62
圖7. vasostatin 於抑制內皮細胞增殖的劑量效應。63
圖8. vasostatin與endostatin對於抑制內皮細胞之劑量效應研究。64
圖9. 內皮細胞株BAEC細胞經過vasostatin (1 mg/ml)處理後不同時間點的形態變化。65
圖10. vasostatin於抑制內皮細胞株BAEC之時間點效應。66
圖11. vasostatin處理纖維母細胞 (3T3 cells)之形態變化。67
圖12. vasostatin 對於纖維母細胞 (3T3 cells)增殖的劑量效應。68
圖13. vasostatin於雞胚胎絨毛膜之新生血管效應研究。69
圖14. vasostatin於抑制雞胚之死亡率。70
圖15. vasostatin抑制帶有腫瘤之老鼠的生長。71
圖16. vasostatin於腫瘤老鼠治療情形。72
圖17.vasostatin於抑制老鼠腫瘤生長之老鼠存活曲線。73
圖18. vasostatin於BAEC處理48小時後之細胞週期分析。75
圖19. Hoechst 33258染色分析,經由vasostatin處理後之BAEC,細胞核變化。76
圖20. vasostatin 對內皮細胞移行之效應研究。77
圖21. vasostatin於內皮細胞移行之劑量效應統計圖。78
圖22. vasostatin對於內皮細胞分泌MMP之效應研究。79
圖23. vasostatin對於內皮細胞其Bcl-2和Bax的西方轉漬圖。80
圖24. vasostatin於抑制內皮細胞 (BAEC)之效應研究。81
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