臺灣博碩士論文加值系統

摘要
血紅素(hemoglobin)存在於紅血球中，為一具有四聚體結構的物質，是血液中輸送氧氣、二氧化碳與氫離子的主要物質。直接以血紅素做為人工替代血液會有以下兩個缺點：(1)血紅素離開紅血球後由於缺乏2,3-DPG的調控，導致血紅素對氧的親和力過高，而降低了其在組織器官的釋氧功能；(2)血紅素的體積太小易經由微血管間隙滲漏出去，導致體內的半衰期太短(約1.5小時)。為了克服血紅素對氧親和力過高的問題，Benesch (1972)首先以Pyridoxyl 5’-phosphate內部交聯血紅素(PLP-Hb)以修飾血紅素對氧的親合力，到了1982年de Venuto and Zegna 進一步利用外部交聯劑來交聯聚合血紅素，提高血紅素的分子量，如此可以有效地增加其半衰期6至7倍。
目前臨床上最常使用的外部交聯劑為glutaraldehyde (GA)，聚合血紅素最重要的就是控制其分子量分佈及適當的攜氧能力，較適當的分子量大小約在200∼400 kdal，以不超過500 kdal為佳，也就是相當於2至8個血紅素分子聚合的大小。若聚合程度過高，則聚合後的血紅素溶液黏度會過大，導致血液流變性質的改變。若血紅素分子聚合程度過低，則無法得到適當的半衰期。然而GA對血紅素分子進行的聚合反應很快，所製造出來的聚合血紅素往往分子量分佈相當廣，容易造成許多過聚合的高分子聚合物(分子量 > 500 kdal)。並且GA聚合血紅素無法在儲存及加熱過程中維持穩定結構，容易釋放出對人體有害的GA分子，因此GA並非製造聚合血紅素最佳的交聯劑。為了克服上述GA交聯血紅素的問題，本研究使用了兩種本實驗室所開發出來的天然交聯劑(genipin與reuterin)，來交聯聚合血紅素做為人工替代血液。
在第一部份的實驗，我們使用一種從中藥梔子果實萃取純化出來的天交聯劑genipin (GP)來交聯血紅素。根據我們先期的體外實驗結果，已證實了天然交聯劑GP的細胞毒性，與目前常使用的化學交聯劑GA比較起來，要明顯低的很多。再加上GP與胺基的反應速率較GA要來的溫和許多，因此在聚合過程中較有利於分子量分佈的控制。實驗主要是尋找出以GP聚合PLP-Hb製備GP-PLP-Hb的適當反應條件，並探討終止聚合反應及移除未聚合PLP-Hb的方法，最後以動物實驗評估在30%及50%血液替代度下老鼠的存活情形，以及在50%血液替代度下GP-PLP-Hb的體內半衰期量測，實驗的對照組為未聚合的血紅素。
體外實驗結果顯示，以GP聚合血紅素確實較GA來得溫和且易於控制聚合度，利用glycine能有效的達到終止聚合血紅素反應的目的。且經由凝膠層析管柱可將未聚合的血紅素完全移除。動物實驗結果顯示，經由未聚合血紅素及GP-PLP-Hb進行30%血液替代時，老鼠均能成功的存活下來，而當血液替代度達到50%時，以未聚合血紅素進行血液替換的老鼠只有一隻成功的存活下來(n = 6)，而以GP-PLP-Hb進行血液替換的老鼠則都成功的存活下來(n = 6)。由此可得知經GP聚合後的血紅素，確實能有效提昇老鼠的存活率。而在50%血液替代度下半衰期的實驗結果顯示，經GP聚合的血紅素能將半衰期由原先未聚合時的1.5小時提昇到12.5小時。
雖然以GP聚合血紅素解決了GA交聯血紅素兩項主要的問題，但是由於血紅素在經由GP聚合後會使得血紅素呈現藍紫色，在感官上可能會有些負面印象，因此在下個部份的實驗，我們使用了本實驗室所開發出來的另一種天然交聯劑reuterin (RR)，來聚合血紅素做為人工替代血液。在這部份的實驗，主要是找出以RR聚合PLP-Hb，製備RR-PLP-Hb的適當反應條件，並探討終止聚合反應及移除未聚合PLP-Hb的方法。由於交聯聚合反應可能會造成血紅素的變性，因此在這部份的實驗同時探討了添加抗氧化劑(維生素C, glutathione以及glucose)，對防止血紅素變性能力的影響，以找出適當的添加濃度，防止聚合過程中變性血紅素的生成。最後以動物實驗的方式，來評估經RR-PLP-Hb進行30%及50%血液替代度後老鼠的存活率，以及在50%血液替代度下其體內半衰期的量測。
體外實驗結果顯示，經由reuterin聚合後的血紅素並不會造成顏色的改變，且RR聚合PLP-Hb的反應速率溫和易於控制聚合血紅素分子量。經由glycine能有效的達到終止聚合反應的目的，同時經由凝膠層析管柱能有效的將未聚合的血紅素移除掉。在防止變性血紅素生成的實驗方面顯示，當血紅素濃度為10 g/dL的情形下，加入300 ppm維它命C, 1250 ppm glutathione及1000 ppm 的glucose時候能有效的防止變性血紅素的生成。在動物實驗結果顯示，經由RR聚合後的血紅素確實能有效的提昇50%血液替代度下老鼠的存活率。且能將體內半衰期由原先未聚合時的1.5小時提昇到12小時。這個結果與以 GP聚合的血紅素相似。
由GP與RR此兩種聚合型的人工替代血液的實驗結果顯示，以GP與RR聚合血紅素做為人工替代血液應是相當可行的。但由於受限於聚合度的關係，聚合後的血紅素仍然有機會經由微血管的間隙滲漏出去，因而使得半衰期有一定的瓶頸存在。因此要更有效的提昇半衰期，必須要增加血紅素的體積。
在最後一部份的實驗，我們探討了使用egg phosphatidylcholine (EPC)及小分子量chitosan製備奈米微粒包覆血紅素的較適製備條件，以增加血紅素的體積。實驗結果顯示，製備出來的微粒粒徑在100-200 nm之間，對血紅素的包覆率約為40%左右，另外經由SEM及TEM的觀察結果得知，此微粒為一具脂雙層結構，且為一表面光滑的球體。

ABSTRACT
Hemoglobin has been used as raw materials for manufacturing blood substitutes. However, because of its high oxygen affinity and short vascular retention time, limitations on hemoglobin as a blood substitute in clinical therapy have been reported in the literature. To decrease its oxygen affinity, hemoglobin has been modified by pyridoxylation (PLP-Hb) and followed by polymerization with glutaraldehyde. It was reported that the polymerized hemoglobin showed a P50 value of 19 to 22 mm Hg. Nevertheless, the reaction rate of hemoglobin with glutaraldehyde is too fast to control its molecular weight distribution. Additionally, the glutaraldehyde-polymerized hemoglobin is relatively unstable and may release glutaraldehyde residues during storage or sterilization. It was reported that glutaraldehyde is cytotoxic even at low doses. This may impair the biocompatibility of the polymerized products.
In an attempt to overcome the aforementioned problems, two naturally occurring crosslinking agents, genipin and reuterin, were used by our group to polymerize hemoglobin. The first study was to investigate the feasibility of using genipin to polymerize hemoglobin as a blood substitute. The results indicated that the rate of hemoglobin polymerization by glutaraldehyde was significantly faster than that by genipin and it readily produced polymers with molecular masses greater than 500,000 daltons. It was found that the maximum degree of hemoglobin polymerization by genipin was approximately 40% if over-polymerization is to be prevented. With increasing the reaction temperature, hemoglobin concentration, and genipin-to-hemoglobin molar ratio, the duration taken to achieve the maximum degree of hemoglobin polymerization by genipin became significantly shorter. The P50 value of the unmodified hemoglobin was 9 mm Hg, while that of the genipin-polymerized PLP-hemoglobin increased to 21 mm Hg. It was found in a rat model that the genipin-polymerized PLP-hemoglobin increased the survive ratio of rats in a 50% blood exchange. The half-life of the unpolymerized hemoglobin in circulation was about 1.5 h, while that of the genipin-polymerized PLP-hemoglobin was approximately 12.5 h.
In the second study, we used another naturally occurring crosslinking agent, reuterin, to polymerize hemoglobin. The results indicated that the rate of hemoglobin polymerization by reuterin was significantly slower than that by glutaraldehyde. In an animal study, it was found that animals transfused with the reuterin-polymerized PLP-hemoglobin up to a 50% blood exchange all survived (n = 6), while animals transfused with allograft plasma died in 3~5 h after transfusion (n = 6) and those transfused with phosphate buffered saline (pH 7.4, n = 6) and unpolymerized hemoglobin (n = 6) survived one out of six. The half-life of the unpolymerized hemoglobin in circulation was about 1.5 h, while that of the reuterin-polymerized hemoglobin was approximately 12 h.
In conclusion, genipin and reuterin are promising agents to polymerize hemoglobin as blood substitutes.

目 錄
內容 頁數
摘要 I
英文摘要 IV
目錄 VI
圖索引 X
表索引 XIV
第一章 研究背景及目的
1.1 人工替代血液的研究方向 1
1.2 碳氟化物 1
1.3 經化學修飾的血紅素 2
1.3.1 包覆型人工替代血液（hemosome） 5
1.3.2 基因重組型人工替代血液（recombinant hemoglobin） 5
1.3.3 聚合型人工替代血液（polymerized hemoglobin） 6
1.3.4 共軛交聯型血紅素（conjugated hemoglobin） 8
1.4 研究動機與目的 9
第二章 以Genipin聚合血紅素做為人工替代血液的體外性質評估
2.1 研究目的 11
2.2 材料與方法 11
2.2.1 豬血紅素的萃取純化 11
2.2.2 血紅素純度鑑定 13
2.2.3 血紅素濃度測量 14
2.2.4 變性血紅素(metHb)含量測定 14
2.2.5 聚合血紅素 16
2.2.6 終止聚合反應方法探討 19
2.2.7 聚合血紅素分離 20
2.2.7.1 離子交換管柱 21
2.2.7.2 凝膠層析管柱 21
2.2.8 氧離曲線測試 21
2.3 實驗結果與討論 23
2.3.1 豬血紅素的萃取純化 23
2.3.2 血紅素聚合條件探討 27
2.3.3 終止聚合反應實驗結果 32
2.3.4 移除未聚合血紅素 38
2.4 結論 42
第三章 以Genipin聚合血紅素做為人工替代血液體內性質評估
3.1 研究目的 43
3.2 材料與方法 43
3.2.1 鈉、鉀離子量測 43
3.2.2 粒徑量測 44
3.2.3 pH值及滲透壓量測 44
3.2.4 存活率測試 44
3.2.5 體內半衰期量測 45
3.3 實驗結果與討論 48
3.3.1 體外各項性質量測 48
3.3.2 存活率測試 50
3.3.3 體內半衰期量測 50
3.4 結論 54
第四章 以Reuterin聚合血紅素做為人工替代血液的體外實驗評估
4.1 研究目的 56
4.2 材料與方法 56
4.2.1 Reuterin的製備 56
4.2.2 Reuterin的分離純化 56
4.2.3 血紅素聚合條件探討 58
4.2.4 終止聚合反應條件探討 58
4.3 實驗結果與討論 59
4.3.1 血紅素聚合條件探討 59
4.3.2 終止聚合反應條件探討 63
4.3.3 移除未聚合血紅素 63
4.4 結論 67
第五章 防止變性血紅素生成的方法
5.1 研究目的 68
5.2 材料與方法 68
5.2.1 維生素C防止血紅素變性能力探討 70
5.2.2 glutathione及glucose防止血紅素變性能力探討 73
5.3 實驗結果與討論 73
5.3.1 維生素C抗氧化能力探討 73
5.3.2 glutathione及glucose對防止血紅素變性的影響 73
5.4 結論 76
第六章 以Reuterin聚合血紅素做為人工替代血液體內性質評估
6.1 研究目的 77
6.2 材料與方法 77
6.2.1 RR-PLP-Hb各項性質量測 77
6.2.2 存活率測試 77
6.2.3 體內半衰期量測 77
6.3 實驗結果 78
6.3.1 體外性質量測 78
6.3.2 存活率測試 79
6.3.3 體內半衰期量測 79
6.4 結論 81
第七章 製備奈米微粒包覆血紅素較適條件探討
7.1 研究目的 82
7.2 材料與方法 82
7.2.1 磷脂質 82
7.2.2 幾丁聚醣(chitosan) 84
7.2.3 製備小分子量chitosan 85
7.2.4 製備具pH敏感性奈米微粒 85
7.2.4.1 製備奈米微粒條件探討 88
7.3 實驗結果與討論 89
7.3.1 小分子量chitosan 89
7.3.2 奈米微粒製備條件探討 90
7.3.2.1 不同振盪時間對微粒粒徑及包覆率的影響 90
7.3.2.2 不同EPC/chitosan重量比對微粒粒徑及包覆
率的影響 93
7.3.2.3 不同EPC濃度對微粒粒徑及包覆率的影響 95
7.4 結論 99
第八章 總結 100
參考文獻 102
著作目錄 113

參考文獻
1. De Venuto, F., Friedman, H.I., Neville, J.R., Peck, C.C., “Appraisal of hemoglobin solution as a blood substitute,” Surgery, Gynecology & Obstetrics, 149, 417-436, 1979.
2. Gould, S.A., Rosen, A.L., Sehgal, L., Sehgal, H., Rice, C.L., “ Hemoglobin solutions as red cell substitutes ,” Trans. Am. Soc. Art. Intern. Organs., 26, 350-353, 1980.
3. Everse, J., Hsia, N., “The toxicity of native and modified hemoglobins,” Free Radical Biology & Medicine, 22, 1075-1099, 1997.
4. Chang, T.M.S., “Future prospects for artificial blood,” Tibtech, 17, 61-67, 1999.
5. Gibson, J.B., Maxwell, R.A., Schweitzer, J.B., Fabian, T.C., Proctor, K.G., “Resuscitation from severe hemorrhagic shock after traumatic brain injury using saline, shed blood, or a blood substitute,” Shock. , 17, 234-244, 2002.
6. Sprung, J., Popp, H., O''Hara, P., Woletz, J., “The successful use of hemoglobin-based oxygen carrier as a primary blood substitute during abdominal aneurysm repair with large blood loss,” Anesthesia & Analgesia , 92, 1413-1415, 2001.
7. Cuignet, O.Y., Baele, P.M., Van-Obbergh, L.J., “A second-generation blood substitute (perflubron emulsion) increases the blood solubility of modern volatile anesthetics in vitro,” Anesthesia & Analgesia , 95, 368-372, 2002.
8. Klein, H.G., “Blood substitutes: how close to a solution? ” Oncology (Huntington), 16, 147-151, 2002.
9. Chang, T.M.S., “Red blood cell substitutes, ” Bailliere''s Best Practice in Clinical Haematology, 13, 651-667, 2000.
10. Obraztsov, V.V., Neslund, G.G., Kornbrust, E.S., Flaim, S.F., Woods, C. M., “In vitro cellular effects of perfluorochemicals correlate with their lipid solubility,” American Journal of Physiology - Lung Cellular & Molecular Physiology , 278, L1018-1024, 2000.
11. Lowe, K.C., “Perfluorinated blood substitutes and artificial oxygen carriers,” Blood Reviews, 13, 171-184, 1999.
12. Lowe, K.C., Anthony, P., Wardrop, J., Davey, M.R., Power, J.B., “Perfluorochemicals and cell biotechnology,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 25, 261-274, 1997.
13. Zuck, T.F., Riess, J.G., “Current status of injectable oxygen carriers,” Critical Reviews in Clinical Laboratory Sciences, 31, 295-324, 1994.
14. Matta, M.S., Willbraham AC., “General, Organic, and Biological Chemistry, 2nd Edition.”
15. 曾國輝, “大學生物化學,” 藝軒書局, 台北, 45-86, 1993.
16. 卓貴美, “圖解生理學,” 五南書局, 台北, 124-159, 2000.
17. Bruce, A.K., Martin, C.H., Steven, H.L., Theodore, W.R., “Effects of tween 80 and sucrose on acute short-term stability and long-term storage at —20℃ of a recombinant hemoglobin,” Journal of Pharmaceutical Sciences, 87, 1062-1068, 1998.
18. Brantley, R.E., Smerdon, S.J., Wilkinson, A.J., Singleton, E.W., Olson, J.S., “The mechanism of autoxidation of myoglobin,” J. Biol. Chem., 268, 6995-7010, 1993.
19. Kan, P., Chen, W.K., Lee, C.J., “Simulation of oxygen saturation of hemoglobin solution, RBC suspension and hemosome by a neural network system,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 24, 143-151, 1996.
20. Wang, Y.C., Lee, C.J., Chen, W. K., Huang , C.I., Chen, W.F., Chen, G.J., Lin, S.Z., “Alteration of cerebral microcirculation by hemodilution with hemosome in awake rats,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 24, 35-42, 1996.
21. Chung, T.W., Chu, S.N., Chen, W.K., Lee, C.J., “A rheological equation to express the relations among hemoglobin contents, hematocrits, and viscosity of hemosome,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 23, 153-161, 1995.
22. Rudolph, A.S., Sulpizio, A., Hieble, P., MacDonald, V., Chavez, M., Feuerstein, G., “ Liposome encapsulation attenuates hemoglobin-induced vasoconstriction in rabbit arterial segments, ” Journal of Applied Physiology, 82, 1826-1835, 1997.
23. Lesile, S.B., Puvvada, S., Ratna, B.R., Rudolph, A.S., “Encapsulation of hemoglobin in a bicontinuous cubic phase lipid,” Biochimica et Biophysica Acta, 1285, 246-254, 1996.
24. Vandegriff, K.D., Wallach, D.F., Winslow, R.M., “Encapsulation of hemoglobin in non-phospholipid vesicles,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 22, 849-854, 1994.
25. Rosado-Ruiz, T., Antommattei-Perez, F.M., Cadilla, C.L., Lopez-Garriga, J., “Expression and purification of recombinant hemoglobin I from Lucina pectinata,” , Journal of Protein Chemistry, 20, 311-315, 2001.
26. Tsai, C.H., Fang, T.Y., Ho, N.T., Ho, C., “Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation,” Biochemistry, 39, 13719-13729, 2000.
27. Devenuto, F., Zegna, A., “Preparation and evaluation of pyridoxylated-polymerized human hemoglobin,” Journal of Surgical Research, 34, 205-212, 1982.
28. Keipert, P.E., Adeniran, A.J., Kwong, S., Benesch, R.E., “Functional properties of a new crosslinked hemoglobin designed for use as a red cell substitute,” 29, 768-773, 1989.
29. Shibayama, N., Imai, K., Hirata, H., Hiraiwn, H., Morimoto, H., Saigel, S., “Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82β1and 82β2 lysyl residues by bis(3,5-dibromosalicyl) fumarate,” Biochemistry, 30, 8158-8165, 1991.
30. Nelson, D., Srnak, A., Ebeling, A., Kunas, G., Catarello, J., Burhop, K., “Synthesis and properties of polymerized diasprin cross-linked hemoglobins,” Biomater. artif. cells immobil. Biotechnol., 20, 253-258, 1992.
31. Kluger, R., Wodzinska, J., Jones, R.T., Head, C., Fujita, T.S., Shih, D.T., “Three-point cross-linking: potential red cell substitutes from the reaction of trimesoyl tris (methyl phosphate) with hemoglobin,” Biochemistry, 31, 7551-7559, 1992.
32. Sehgal, L.R., Rosen, A.L., Gould, S.A., Sehgal, H.L., Moss, G.S., “Preparation and in vitro characteristics of polymerized pyridoxylated hemoglobin,” Transfusion, 23, 158-162, 1983.
33. Marini, M.A., Moore, G.L., Christensen, S.M., Fishman, R.M., Jessee, R.G., Medina, F., Snell, S.M., Zegna, A.I., “Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde,” Biopolymers, 29, 871-882, 1990.
34. Feola, M., Gonzalez, H., Canizaro, P.C., Bingham, D., Periman, P., “Development of a bovine stroma-free hemoglobin solution as a blood substitute,” Surgery gynecology & obstetrics, 157, 399-408, 1983.
35. Lee, R., Atsumi, N., Jacobs, E.E, Gerald, A.W., Vlahakes, G.J., “Ultrapure, stroma-free, polymerized bovine hemoglobin solution: evaluation of renal toxicity,” Jouranl of surgical research, 47, 407-411, 1989.
36. Hu, T., Su, Z., “Preparation of well-defined bovine polyhemoglobin based on dimethyl adipimidate and glutaraldebyde cross-linkage,” Biochemical & Biophysical Research Communications. 293, 958-961, 2002.
37. Alayash, A.I., Summers, A.G., Wood, F.J.Y., “Effects of glutaraldehyde polymerization on oxygen transport and redox properties of bovine hemoglobin,” Archives of Biochemistry and Biophysics, 391, 225-34, 2001.
38. Bakker, J.C., Berbers, A.M., Bleeker, W.K., Boer, P.J., Biessel, T.M., “Preparation and characterization of crosslinked and polymerized hemoglobin solution,” Biomater. artif. cells immobil. Biotechnol., 20, 233-241, 1992.
39. Tsai, S.P., Wong, J.T.F., “Enhancement of erythrocyte sedimentation rate by polymerized hemoglobin,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 24, 513-523, 1996.
40. MacDonald, S.L., Pepper, D.S., “Hemoglobin polymerization,” Methods in Enzymology, Everse, J., Vandegriff, K.D., Winslow, R.M., 231, 287-308, 1994.
41. Gould, S.A., Moore, E.E., Hoyt, D.B., Ness, P.M., Norris, E.J., Carson, J.L., Hides, G.A., Freeman, I.H., DeWoskin, R., Moss, G.S., “The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable,” Journal of the American College of Surgeons. 195, 445-452, discussion 452-455, 2002.
42. Leytin, V., Mazer, D., Mody, M., Garvey, B., Freedman, J., “Hemolink, an o-raffinose cross-linked haemoglobin-based oxygen carrier, does not affect activation and function of human platelets in whole blood in vitro,” British Journal of Haematology, 120, 535-541, 2003.
43. Ortegon, D.P., Davis, M.R., Dixon, P.S., Smith, D.L., Josephs, J.D., Mueller, D.L., Jenkins, D.H., Kerby, J.D., “The polymerized bovine hemoglobin-based oxygen-carrying solution (HBOC-201) is not toxic to neural cells in culture,” Journal of Trauma-Injury Infection & Critical Care, 53, 1068-1072, 2002.
44. Okayama, N., Park, J.H., Coe, L., Granger, D.N., Ma, L., Hisa, C.J., Alexander, J.S., “Polynitroxyl alphaalpha-hemoglobin (PNH) inhibits peroxide and superoxide-mediated neutrophil adherence to human endothelial cells,” Free Radical Researc., 31, 53-58, 1999.
45. Caron, A., Malfatti, E., Aguejouf, O., Faivre-Fiorina, B., Menu, P., “Vasoconstrictive response of rat mesenteric arterioles following infusion of cross-linked, polymerized, and conjugated hemoglobin solutions,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 29, 19-30, 2001.
46. Conover, C.D., Linberg, R., Shum, K.L., Shorr, R.G., “The ability of polyethylene glycol conjugated bovine hemoglobin (PEG-Hb) to adequately deliver oxygen in both exchange transfusion and top-loaded rat models,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 27, 93-107, 1999.
47. Conover, C.D., Linberg, R., Gilbert, C.W., Shum, K.L., Shorr, R.G., “Effect of polyethylene glycol conjugated bovine hemoglobin in both top-load and exchange transfusion rat models,” Artificial Organs, 21, 1066-1075, 1997.
48. Caron, A., Menu, P., Faivre-Fiorina, B., Labrude, P., Vigneron, C., “The effects of stroma-free and dextran-conjugated hemoglobin on hemodynamics and carotid blood flow in hemorrhaged guinea pigs,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 27, 49-64, 1999.
49. Quellec, P., Leonard, M., Grandgeorge, M., Dellacherie, E., “Human hemoglobin conjugated to carboxylate dextran as a potential red blood cell substitute. I. Further physico-chemical characterization,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 22, 669-676, 1994.
50. Caron, A., Malfatti, E., Aguejouf, O., Faivre-Fiorina, B., Menu, P., “Vasoconstrictive response of rat mesenteric arterioles following infusion of cross-linked, polymerized, and conjugated hemoglobin solutions,” Artificial Cells, Blood Substitutes, & Immobilization Biotechnology, 29, 19-30, 2001.
51. Veronese, F.M., Morpurgo, M., “Bioconjugation in pharmaceutical chemistry,” Farmaco, 54, 497-516, 1999.
52. Sakai, H., Yuasa, M., Onuma, H., Takeoka, S., Tsuchida, E., “Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: objective comparison between cellular and acellular types,” Bioconjugate Chemistry, 11, 56-64, 2000.
53. Sung, H.W., Huang, R.N., Huang, L.L.H., Tsai, C.C., “ In Vitro Evalualtion of Cytotoxicity of a Naturally Occurring Crosslinking Reagent for Biological Tissue Fixation,” Journal of Biomaterials Science-Polymer Edition, 13, 63-78, 1998.
54. Tsai, C.C., Huang, R.N., Huang, L.L.H., Lin, C.K., Chen, C.N., Sung, H.W., “A Natural Crosslinking Reagent for Biological Tissue Fixation: An In Vitro Cytotoxicity Study, ” R.O.C. & Japan Joint Symposium on Biomaterials and Artificial Organs, 153-155, 1998.
55. Darin, S.K., Steven, P.W., Wen, H., Ramesh, K., David, W.C., “Structure determination of aquomet procine hemoglobin at 2.8Å resolution,” Journal of Molecular Biology, 224, 541-553, 1994.
56. Hart, R.A., Baiely, J.E., “Purificiation and aqueous two-phase partitioning properties of recombinant vitreoscilla hemoglobin,” Enzyme microb. Technol., 13, 788-795, 1991.
57. Russell, M.C., Lawson, Q., “Analysis of protein association by partitioning in aqueous two-phase polymer system: application to the tetramer-dimer dissociation of hemoglobin,” Analytical Biochemistry, 105, 99364-368, 1980.
58. Chen, C.N., Liang, H.F., Lin, M.H., Sung, H.W., “A Natural Sterilant (Reuterin) Fermented from Glycerol Using Lactobacillus Reuteri: Fermentation Conditions,” Chinese Journal of Medical and Biological Engineering, 21, 205-212, 2001.
59. Lie-Injo, L.E., Solai, A., Ganesan, J., “Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains,” Journal of Chromatography A., 153, 161-165, 1978.
60. Crosby, W.H., Munn, J.I., Furth, F.W., “Standardizing a method for clinical hemoglobinometry,” U.S. Armed. Forces Med. J., 5, 693, 1954.
61. Toshiyasu, M., “Determination of Methemoglobin and Carboxyhemoglobin in Blood by Rapid Colorimetry,” Biol. Pharm. Bull., 20, 1208-1211, 1997.
62. Kilmartin, J.V., “Preparation and characterization of Modified Hemoglobins,” Methods in Enzymology, Antonini, E., Rossi-Bernardi, L., Chiancone, E., 76, 147, 1981.
63. Neville, J.R., “Hemoglobin oxygen affinity measurement using biotonometry,” J. Appl. Physiol., 37, 967-971, 1974.
64. Bleeker, W., Van der Plas, J., Feitsma, R., Agterberg, J., Rigter, G., DeVries-van Rossen, A., Pauwels, E., Bakker, J., “ In vivo distribution and elimination of hemoglobin modified by intramolecular crosslinking with 2-nor-2-formylpyridoxal5’-phosphate,” J. Lab. Clin. Med., 113, 151-161, 1989.
65. Ning, J., Peterson, L., Anderson, P., Biro, G., “Systemic hemodynamic and renal effects of unmodified human SFHS in dogs,” Biomat. Art. Cells, Immob. Biotech., 20, 723-727, 1992.
66. Lenz, G., Junger, H., Scheider, M., Kothe, N., Lissner, R., Prince, A., “ Elimination of pyridoxylated polyhemoglobin after partial exchange transfusion in chimpanzees,” Biomat. Art. Cells, Immob. Biotech., 19, 699-708, 1991.
67. Savitsky, J., Doczi, J., Black, J., Arnold, J., “A clinical safety trial of stroma-free hemoglobin,” Clin. Pharmacol. Ther., 23, 73-80, 1978.
68. Bunn, H.F., Esham, W.T., Bull, R.W., “The renal handling of hemoglobin. I. Glomerular filtration,” J. Exp. Med., 129, 909-923, 1969.
69. 卓貴美, “圖解生理學,” 五南書局, 台北,217-218, 2000.
70. http://www.nobel.se/medicine/laureates/1998/illpres/enzyme.html
71. Goda, N., Suzuki, K., Naito, M., Takeoka, S., Tsuchida, E., Ishimura, Y., Tamatani, T., Suematsu, M., “Distribution of heme oxygenase isoforms in rat liver: topographic basis for carbon monoxide-mediated microvascular relaxation,” J. Clin. Invest., 101, 604-612, 1998.
72. Talarico, T.L., Casas, I.A., Chung, T.C., Dobrogosz, W.J., “Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri, ” Antimicrobial Agents and Chemitherapy, 1854-1858, 1988.
73. Pincemal, J., Detry, O., Philippart, C., Defraigne, J. O., Franssen, C., Burhop, K., Deby, C., Meurisse, M., Lamy, M., “Diasoirin crosslinked hemoglobin (DCLHBTM): absence of increased free radical generation following administration in a rabbit model of renal ischemia and reperfusion,” Free radical biology & Medicine, 19, 1-9, 1995.
74. 周昆達等編譯, “Stryer’s生物化學2”, Fourth Edition, 合記圖書出版社,台北, 568.
75. 周昆達等編譯, “Stryer’s生物化學1”, Fourth Edition, 合記圖書出版社,台北, 455.
76. 周昆達等編譯, “Stryer’s生物化學2”, Fourth Edition, 合記圖書出版社,台北, 466.
77. Patel, H.M., “Serum opsonins and liposomes - their interaction and opsonophagocytosis,” Critical Reviews in Therapeutic Drug Carrier Systems, 9, 39-90, 1992.
78. Semple, S.C., Chonn, A., Cullis, P.R., “Influence of cholesterol on the association of plasma proteins with liposomes,” Biochemistry, 35, 2521-2525, 1996.
79. Woodle, M.C., Lasic, D.D., “Sterically stabilized liposomes,” Biochim. Biophys. Acta., 1113, 171-199, 1992.
80. 周昆達等編譯, “Stryer’s生物化學2”, Fourth Edition, 合記圖書出版社,台北, 265-266.
81. http://www.chem.ucla.edu/dept/Faculty/hawthorne/Liposomes/liposomes.html