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研究生:錢信榮
研究生(外文):CHIEN HSIN-JUNG
論文名稱:血液凋亡小體收集方法之改良與其在癌症檢測上之應用
論文名稱(外文):Improvement of the Method for Collecting Blood Apoptotic Bodies for Cancer Diagnosis
指導教授:邱全芊
指導教授(外文):CHIOU CHIUAN-CHIAN
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:38
中文關鍵詞:凋亡小體
外文關鍵詞:Apoptotic bodies
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當人體發生疾病或受到傷害時通常會導致細胞的凋亡,並釋出凋亡小體進入血液循環中。因此,這些游離凋亡小體帶著許多有用的訊息(例如:特異性蛋白質或基因的表現)可以作為疾病診斷之用。本實驗室先前已經發展出一個收集游離凋亡小體的方法並証明凋亡小體中測得疾病特異RNA,該方法是利用annexin A5接到磁珠上使磁珠可以與凋亡小體結合,本研究的目的在進一步改良這個方法,使其可以使用於醫學研究或臨床檢測上。首先,我們發現增加RNA萃取的步驟可以改善先前RT-PCR效率不佳的情形。在含有15 mM鈣離子的環境下,2 mg的磁珠約可結合2.2 µg protein的凋亡小體,這樣的磁珠量可以回收溶液中60%的凋亡小體。接著,我們測試抗凝血劑對磁珠捕捉凋亡小體的影響,發現heparin抗凝血劑會干擾磁珠捕捉凋亡小體或RT-PCR。而以EDTA為抗凝血劑收集的血液經過1600 g離心下得到的血漿可以降低血小板對磁珠的競爭。最後,我們應用此方法收集病人血漿中的凋亡小體,並發現凋亡小體中有癌症特異基因CK19的表現。總結上述結果,我們利用2 mg的annexin A5-磁珠在含有15 mM鈣離子的環境下,可以收集血中的凋亡小體,並可以以RT-PCR分析凋亡小體中的疾病特異基因的表現。我們希望未來可以將這個方法應用於疾病診斷。
Severe diseases or injury usually cause apoptosis of cells, which may release apoptotic bodies into circulation. These circulating apoptotic bodies thus contain valuable information (e.g. the specific protein or gene expression) of the original cells from which they were derived and can be useful for diagnostics. The aim of this study is to develop a convenient procedure to collect circulating apoptotic bodies in which disease-specific mRNA can be detected. First, magnetic beads coated with annexin A5, which bound phosphatidylserine on the surface of apoptotic cells, were tested for their capability of capturing apoptotic bodies. In an optimized buffer (HEPES-buffered saline containing 15 mM CaCl2), up to 60% of apoptotic bodies could be recovered by the magnetic beads from a suspension. Real-time reverse transcription-PCR further proved that specific gene expression can be detected quantitatively from the collected apoptotic bodies. Next, suitable conditions for blood sample preparation were tested. The result showed that blood collected in an EDTA-tube had much higher recovery rate of apoptotic bodies than that collected in a heparin-tube, suggesting that heparin might interfere either the binding of apoptotic bodies to annexin A5 or the subsequent assays. We applied this method to collect apoptotic bodies from cancer patients and found that CK19 mRNA can be detected in patients’ blood. In conclusion, we have developed a magnetic bead-based procedure for collection of circulating apoptotic bodies which might prove to be useful for disease diagnosis.
目錄
指導教授推薦書.........................................................................................
口試委員會審定書.....................................................................................
授權書.......................................................................................................iii
誌謝...........................................................................................................iv
中文摘要....................................................................................................v
英文摘要...................................................................................................vi
縮寫表......................................................................................................vii目錄.........................................................................................................viii
圖表目錄...................................................................................................xi
第一章 文獻回顧......................................................................................1
1-1癌症檢測......................................................................................1
1-2 血液中的游離核酸(cell free nucleic acids) ...............................1
1-2-1 游離DNA.........................................................................2
1-2-2 游離RNA.........................................................................2
1-3 細胞凋亡(apoptosis) ..................................................................2
1-4 細胞膜內外翻轉(cell membrane externaliztion) .......................3
1-5 可以與phosphatidylserine (PS)鍵結的蛋白質..........................4
1-6 Cytokeratin 19 (CK19) ................................................................4
1-7 先前實驗室成果.........................................................................5
第二章 研究目的......................................................................................6
第三章 材料與方法..................................................................................7
3-1藥品..............................................................................................7
3-2 製備接有annexin A5的磁珠 ...................................................7
3-3 細胞培養 ...................................................................................8
3-4 誘導細胞凋亡的藥品 ...............................................................9
3-5 誘導細胞凋亡.............................................................................9
3-6 凋亡小體的定量.........................................................................9
3-7 Annexin A5-磁珠捕捉培養液中的凋亡小…............................10
3-8 Annexin A5-磁珠捕捉血清或血漿中的凋亡小體....................10
3-9 萃取RNA..................................................................................10
3-10 One step real-time RT-PCR......................................................11
3-11 回收率的計算.........................................................................11
3-12 β2M與CK19基因的絕對定量標準曲線..............................12
第四章 結果............................................................................................13
4-1 TRIzol萃取凋亡小體RNA去除磁珠與Ca-HEPES buffer的干擾................................................................................................13
4-2 尋找癌症特異基因CK19、CK20、CEA表現量較高的細胞株...............................................................................................13
4-3 比較兩種市售磁珠捕捉凋亡小體的能力...............................14
4-4 增加磁珠量對凋亡小體回收率的影響...................................14
4-5 鈣離子濃度對凋亡小體回收率的影響...................................15
4-6 測試annexin-A5磁珠的capacity............................................16
4-7 採血管的選擇...........................................................................16
4-8 離心力對血中凋亡小體含量的影響.......................................17
4-9 檢體保存與解凍次數的測試...................................................18
4-10 以磁珠捕捉癌症病人血中的凋亡小體分析CK19的量….19
第五章 討論............................................................................................20
5-1 RNA萃取步驟提高RT-PCR靈敏度.......................................20
5-2 EDTA採血管較適合用於磁珠捕捉血漿游離凋亡.................20
5-3重複冷凍解凍會破壞血液中的凋亡小體................................21
5-4 未來發展...................................................................................21
5-5 結論...........................................................................................22
引用文獻..................................................................................................24
附表.........................................................................................................28
附圖.........................................................................................................29

圖表目錄
表一 Primer及probe序列.......................................................................28
圖一 比較有無RNA萃取步驟對RT-PCR的影響…………………….29
圖二 Β2M、CEA、CK-19、CK-20基因在AGS、Colo-205、 Colo-320、HCT-15、HT-29、LoVo、KATOIII細胞株表現情形…..30
圖三 比較不同廠牌的annexin A5-磁珠回收凋亡小體的效率………31
圖四 Annexin A5-磁珠使用量影響凋亡小體的回收率………………32
圖五 鈣離子濃度對annexin A5-磁珠捕捉凋亡小體的影響…………33
圖六 磁珠對凋亡小體的最大結合力…………………………………34
圖七 比較三種不同採血管對於回收檢體中凋亡小體效率的影響…35
圖八 比較不同離心轉速對去除血小板干擾的影響………………….36
圖九 反覆解凍對凋亡小體的影響…………………………………….37
圖十 比較正常人與癌症病人凋亡小體的CK19表現情形……….…38
1. Perkins GL, Slater ED, Sanders GK, Prichard JG. Serum tumor markers. Am Fam Physician 2003;68:1075-82.
2. Wiedemann LM, Morgan GJ. How are cancer associated genes activated or inactivated? Eur J Cancer 1992;28:248-51.
3. Bustin SA, Mueller R. Real-time reverse transcription PCR and the detection of occult disease in colorectal cancer. Mol Aspects Med 2006;27:192-223.
4. Chan AK, Chiu RW, Lo YM. Cell-free nucleic acids in plasma, serum and urine: a new tool in molecular diagnosis. Ann Clin Biochem 2003;40:122-30.
5. Angert RM, LeShane ES, Lo YM, Chan LY, li-Bovi LC, Bianchi DW. Fetal cell-free plasma DNA concentrations in maternal blood are stable 24 hours after collection: analysis of first- and third-trimester samples. Clin Chem 2003;49:195-8.
6. Wataganara T, Bianchi DW. Fetal cell-free nucleic acids in the maternal circulation: new clinical applications. Ann N Y Acad Sci 2004;1022:90-9.
7. Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994;3:67-71.
8. Skvortsova TE, Rykova EY, Tamkovich SN, Bryzgunova OE, Starikov AV, Kuznetsova NP et al. Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation. Br J Cancer 2006;94:1492-5.
9. Tsui NB, Ng EK, Lo YM. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem 2002;48:1647-53.
10. Reddi KK, Holland JF. Elevated serum ribonuclease in patients with pancreatic cancer. Proc Natl Acad Sci U S A 1976;73:2308-10.
11. Lo KW, Lo YM, Leung SF, Tsang YS, Chan LY, Johnson PJ et al. Analysis of cell-free Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma. Clin Chem 1999;45:1292-4.
12. Kopreski MS, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999;5:1961-5.
13. Ng EK, Tsui NB, Lam NY, Chiu RW, Yu SC, Wong SC et al. Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals. Clin Chem 2002;48:1212-7.
14. El-Hefnawy T, Raja S, Kelly L, Bigbee WL, Kirkwood JM, Luketich JD, Godfrey TE. Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics. Clin Chem 2004;50:564-73.
15. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-95.
16. Daniel PT, Sturm I, Ritschel S, Friedrich K, Dorken B, Bendzko P, Hillebrand T. Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers. Anal Biochem 1999;266:110-5.
17. Halicka HD, Bedner E, Darzynkiewicz Z. Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis. Exp Cell Res 2000;260:248-56.
18. Holdenrieder S, Stieber P. Apoptotic markers in cancer. Clin Biochem 2004;37:605-17.
19. Bretscher MS. Asymmetrical lipid bilayer structure for biological membranes. Nat New Biol 1972;236:11-2.
20. Boersma HH, Kietselaer BL, Stolk LM, Bennaghmouch A, Hofstra L, Narula J et al. Past, present, and future of annexin A5: from protein discovery to clinical applications. J Nucl Med 2005;46:2035-50.
21. Hoffmann K, Kerner C, Wilfert W, Mueller M, Thiery J, Hauss J, Witzigmann H. Detection of disseminated pancreatic cells by amplification of cytokeratin-19 with quantitative RT-PCR in blood, bone marrow and peritoneal lavage of pancreatic carcinoma patients. World J Gastroenterol 2007;13:257-63.
22. Suo J, Wang Q, Jin HJ, Li H, Zhao H. K-19 mRNA RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer. World J Gastroenterol 2006;12:5219-22.
23. Ge MJ, Shi D, Wu QC, Wang M, Li LB. Observation of circulating tumour cells in patients with non-small cell lung cancer by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction in peroperative period. J Cancer Res Clin Oncol 2006;132:248-56.
24. Wang JY, Wu CH, Lu CY, Hsieh JS, Wu DC, Huang SY, Lin SR. Molecular detection of circulating tumor cells in the peripheral blood of patients with colorectal cancer using RT-PCR: significance of the prediction of postoperative metastasis. World J Surg 2006;30:1007-13.
25. Schoenfeld A, Kruger KH, Gomm J, Sinnett HD, Gazet JC, Sacks N et al. The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 1997;33:854-61.
26. Shin-Che Hsu, Chiuan-Chian Chiou. Circulating apoptotic bodies as a target for cancer diagnosis. 長庚大學碩士論文 2005.
27. Sobel M, Fish WR, Toma N, Luo S, Bird K, Mori K et al. Heparin modulates integrin function in human platelets. J Vasc Surg 2001;33:587-94.
28. Jung R, Lubcke C, Wagener C, Neumaier M. Reversal of RT-PCR inhibition observed in heparinized clinical specimens. Biotechniques 1997;23:24, 26, 28.
29. Bai X, Fischer S, Keshavjee S, Liu M. Heparin interference with reverse transcriptase polymerase chain reaction of RNA extracted from lungs after ischemia-reperfusion. Transpl Int 2000;13:146-50.
30. Lentz BR. Exposure of platelet membrane phosphatidylserine regulates blood coagulation. Prog Lipid Res 2003;42:423-38.
31. Borisenko GG, Iverson SL, Ahlberg S, Kagan VE, Fadeel B. Milk fat globule epidermal growth factor 8 (MFG-E8) binds to oxidized phosphatidylserine: implications for macrophage clearance of apoptotic cells. Cell Death Differ 2004;11:943-5.
32. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature 2002;417:182-7.
33. Dasgupta SK, Guchhait P, Thiagarajan P. Lactadherin binding and phosphatidylserine expression on cell surface-comparison with annexin A5. Transl Res 2006;148:19-25.
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