• 西安交通大學(xué)醫(yī)學(xué)院第一附屬醫(yī)院肝膽外科（西安， 710061）;

目的綜述豬胰島細(xì)胞分離純化的常用方法及最新研究進(jìn)展。 方法查閱近年來(lái)國(guó)內(nèi)外關(guān)于豬胰島細(xì)胞分離純化的相關(guān)文獻(xiàn)，進(jìn)行總結(jié)分析。 結(jié)果分離純化的效果主要取決于供體的前期篩選、高質(zhì)量胰腺的獲取與有效保存，以及分離純化方法的選擇與改進(jìn)。 結(jié)論豬胰島分離純化操作的優(yōu)化與標(biāo)準(zhǔn)化的建立，極大促進(jìn)了異種胰島移植研究的發(fā)展，有望解決目前移植供體短缺的問(wèn)題。

引用本文： 朱海濤,于良,王博. 豬胰島細(xì)胞分離純化研究進(jìn)展. 中國(guó)修復(fù)重建外科雜志, 2012, 26(8): 1012-1016. doi: 復(fù)制

1. Kobayashi N. The current status of islet transplantation and its perspectives. Rev Diabet Stud, 2008, 5(3): 136-143.
2. Noguchi H. Pancreatic islet transplantation. World J Gastrointest Surg, 2009, 1(1): 16-20.
3. 趙勇. 異種移植免疫學(xué). 北京: 中國(guó)醫(yī)藥科技出版社, 2003: 141-189.
4. Marigliano M, Bertera S, Grupillo M, et al. Pig-to-nonhuman primates pancreatic islet xenotransplantation: an overview. Curr Diab Rep, 2011, 11(5): 402-412.
5. Hardikar AA, Wang XY, Williams LJ, et al. Functional maturation of fetal porcine β-cells by glucagon-like peptide 1 and cholecystokinin. Endocrinology, 2002, 143(9): 3505-3514.
6. Dufrane D, Gianello P. Pig islets for clinical islet xenotransplantation. Curr Opin Nephrol Hypertens, 2009, 18(6): 495-500.
7. Bennet W, Björkland A, Sundberg B, et al. A comparison of fetal and adult porcine islets with regard to Gal alpha (1, 3)Gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. Transplantation, 2000, 69(8): 1711-1117.
8. Groth CG, Korsgren O, Tibell A, et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet, 1994, 344(8934): 1402-1404.
9. Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med, 2006, 12(3): 304-306.
10. Bottino R, Balamurugan AN, Smetanka C, et al. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies. Xenotransplantation, 2007, 14(1): 74-82.
11. Dufrane D, D’hoore W, Goebbels RM, et al. Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models. Xenotransplantation, 2006, 13(3): 204-214.
12. Rood PP, Buhler LH, Bottino R, et al. Pig-to-nonhuman primate islet xenotransplantation: a review of current problems. Cell Transplant, 2006, 15(2): 89-104.
13. Dufrane D, Goebbels RM, Fdilat I, et al. Impact of porcine islet size on cellular structure and engraftment after transplantation: adult versus young pigs. Pancreas, 2005, 30(2): 138-147.
14. Kim HI, Lee SY, Jin SM, et al. Parameters for successful pig islet isolation as determined using 68 specific-pathogen-free miniature pigs. Xenotransplantation, 2009, 16(1): 11-18.
15. Kim JH, Kim HI, Lee KW, et al. Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs. Xenotransplantation, 2007, 14(1): 60-66.
16. 蔣小峰, 錢(qián)滔來(lái), 向廣陽(yáng), 等. 五指山小型豬胰島分離和純化的初步研究. 臨床醫(yī)學(xué)工程, 2011, 18(9): 1343-1347.
17. Anazawa T, Balamurugan AN, Matsumoto S, et al. Rapid quantitative assessment of the pig pancreas biopsy predicts islet yield. Transplant Proc, 2010, 42(6): 2036-2039.
18. Jin SM, Shin JS, Kim KS, et al. Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation, 2011, 18(6): 369-379.
19. van der Windt DJ, Bottino R, Casu A, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant, 2009, 9(12): 2716-2726.
20. d’Apice AJ, Cowan PJ. Gene-modified pigs. Xenotransplantation, 2008, 15(2): 87-90.
21. Ekser B, Cooper DK. Overcoming the barriers to xenotransplantation: prospects for the future. Expert Rev Clin Immunol, 2010, 6(2): 219-230.
22. Le Bas-Bernardet S, Tillou X, Poirier N, et al. Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons. Transplant Proc, 2011, 43(9): 3426-3430.
23. Goto M, Imura T, Inagaki A, et al. The impact of ischemic stress on the quality of isolated pancreatic islets. Transplant Proc, 2010, 42(6): 2040-2042.
24. 張明, 杜成友. 不同熱缺血時(shí)間對(duì)豬胰島獲取及功能的影響. 中國(guó)組織工程研究與臨床康復(fù), 2011, 15(5): 847-850.
25. Kuroda Y, Fujino Y, Morita A, et al. Oxygenation of the human pancreas during preservation by a two-layer (University of Wisconsin solution/perfluorochemical) cold-storage method. Transplantation, 1992, 54(3): 561-562.
26. Gioviale MC, Damiano G, Palumbo VD, et al. Pancreatic islets from non-heart-beating donor pig: two-layer preservation method in an in vitro porcine model. Int J Artif Organs, 2011, 34(6): 519-525.
27. Brandhorst D, Iken M, Brendel MD, et al. Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas. Transplant Proc, 2005, 37(1): 458-459.
28. Noguchi H, Ueda M, Nakai Y, et al. Modified two-layer preservation method (M-Kyoto/PFC) improves islet yields in islet isolation. Am J Transplant, 2006, 6(3): 496-504.
29. Brandhorst D, Iken M, Brendel MD, et al. Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation. Transplant Proc, 2005, 37(1): 229-230.
30. Taylor MJ, Baicu S, Greene E, et al. Islet isolation from juvenile porcine pancreas after 24-h hypothermic machine perfusion preservation. Cell Transplant, 2010, 19(5): 613-628.
31. 王玨, 侯宗柳, 李汝紅. 半自動(dòng)純化法分離純化成年豬胰島細(xì)胞的實(shí)驗(yàn)研究. 昆明醫(yī)學(xué)院學(xué)報(bào), 2011, 32(10): 14-18.
32. Yonekawa Y, Matsumoto S, Okitsu T, et al. Effective islet isolation method with extremely high islet yields from adult pigs. Cell Transplant, 2005, 14(10): 757-762.
33. Gray DW, Sudhakaran N, Titus TT, et al. Development of a novel digestion chamber for human and porcine islet isolation. Transplant Proc, 2004, 36(4): 1135-1138.
34. Kim SC, Han DJ, Kang CH, et al. Experimental islet isolation in porcine pancreas with new enzyme Liberase PI. Transplant Proc, 2004, 36(7): 2197-2199.
35. Bucher P, Bosco D, Mathe Z, et al. Optimization of neutral protease to collagenase activity ratio for islet of Langerhans isolation. Transplant Proc, 2004, 36(4): 1145-1146.
36. Brandhorst D, Huettler S, Alt A, et al. Adjustment of the ratio between collagenase class II and I improves islet isolation outcome. Transplant Proc, 2005, 37(8): 3450-3451.
37. Brandhorst H, Alt A, Huettler S, et al. The ratio between class II and class I collagenase determines the amount of neutral protease activity required for efficient islet release from the rat pancreas. Transplant Proc, 2005, 37(1): 215-216.
38. Hilling DE, Rijkelijkhuizen JK, Töns HA, et al. Porcine islet isolation outcome is not affected by the amount and distribution of collagen in the pancreas. Xenotransplantation, 2010, 17(3): 250-255.
39. Kinasiewicz A, Juszczak M, Pachecka J, et al. Pancreatic islets isolation using different protocols with in situ flushing and intraductal collagenase injection. Physiol Res, 2004, 53(3): 327-333.
40. Andrades P, Asiedu C, Ray P, et al. Islet yield after different methods of pancreatic liberase delivery. Transplant Proc, 2007, 39(1): 183-184.
41. Szot GL, Koudria P, Bluestone JA. Murine pancreatic islet isolation. J Vis Exp, 2007, (7): 255.
42. Atwater I, Guajardo M, Caviedes P, et al. Isolation of viable porcine islets by selective osmotic shock without enzymatic digestion. Transplant Proc, 2010, 42(1): 381-386.
43. 張偉杰, Schre J. 成年豬胰島的分離與純化. 中華器官移植雜志, 2000, 21(6): 341-343.
44. Qiao AY, Zhang WH, Chen XJ, et al. Isolation and purification of islet cells from adult pigs. Transplant Proc, 2010, 42(5): 1830-1834.
45. 徐艷艷, 傅紅興, 王浙明, 等. 大鼠胰島分離純化技術(shù)的改良及活性研究. 中國(guó)修復(fù)重建外科雜志, 2010, 24(4): 406-409.
46. 楊學(xué)偉, 章樂(lè)虹, 胡以則, 等. 成年豬胰島細(xì)胞的分離純化及其微囊包裹. 武漢大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2007, 28(7): 496-499.
47. 李鵬, 李汝紅, 趙維山, 等. 云南小耳豬胰島細(xì)胞的分離和純化方法. 昆明醫(yī)學(xué)院學(xué)報(bào), 2009, 30(12): 38-40.
48. Eckhardt T, Jahr H, Federlin K, et al. Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice. J Mol Med (Berl), 1999, 77(1): 123-125.
49. 張樺, 陳宏, 張振, 等. 碘克沙醇及Ficoll-400分離純化大鼠胰島細(xì)胞的效果對(duì)比. 世界華人消化雜志, 2007, 15(15): 1759-1762.
50. 黃躍南, 郭欣, 田素禮, 等. 成年豬胰島分離純化方法的改良. 世界華人消化雜志, 2005, 13(15): 1912-1914.
51. Min T, Yi L, Chao Z, et al. Superiority of visipaque (iodixanol)-controlled density gradient over Ficoll-400 in adult porcine islet purification. Transplant Proc, 2010, 42(5): 1825-1829.
52. 楊卿堯, 田郁文. 胰島細(xì)胞移植. 臺(tái)灣醫(yī)學(xué), 2007, 11(1): 42-46.
53. Shawl AI, Park KH, Kim UH. Insulin receptor signaling for the proliferation of pancreatic β-cells: involvement of Ca2+ second messengers, IP3, NAADP and cADPR. Islets, 2009, 1(3): 216-223.
54. Dekki N, Nilsson R, Norgren S, et al. Type 1 diabetic serum interferes with pancreatic beta-cell Ca2+-handling. Biosci Rep, 2007, 27(6): 321-326.
55. Corbin KL, Hall TE, Haile R, et al. A novel fluorescence imaging approach for comparative measurements of pancreatic islet function in vitro. Islets, 2011, 3(1): 14-20.
56. Elliott RB. Towards xenotransplantation of pig islets in the clinic. Curr Opin Organ Transplant, 2011, 16(2): 195-200.
  1. 1. Kobayashi N. The current status of islet transplantation and its perspectives. Rev Diabet Stud, 2008, 5(3): 136-143.
  2. 2. Noguchi H. Pancreatic islet transplantation. World J Gastrointest Surg, 2009, 1(1): 16-20.
  3. 3. 趙勇. 異種移植免疫學(xué). 北京: 中國(guó)醫(yī)藥科技出版社, 2003: 141-189.
  4. 4. Marigliano M, Bertera S, Grupillo M, et al. Pig-to-nonhuman primates pancreatic islet xenotransplantation: an overview. Curr Diab Rep, 2011, 11(5): 402-412.
  5. 5. Hardikar AA, Wang XY, Williams LJ, et al. Functional maturation of fetal porcine β-cells by glucagon-like peptide 1 and cholecystokinin. Endocrinology, 2002, 143(9): 3505-3514.
  6. 6. Dufrane D, Gianello P. Pig islets for clinical islet xenotransplantation. Curr Opin Nephrol Hypertens, 2009, 18(6): 495-500.
  7. 7. Bennet W, Björkland A, Sundberg B, et al. A comparison of fetal and adult porcine islets with regard to Gal alpha (1, 3)Gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. Transplantation, 2000, 69(8): 1711-1117.
  8. 8. Groth CG, Korsgren O, Tibell A, et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet, 1994, 344(8934): 1402-1404.
  9. 9. Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med, 2006, 12(3): 304-306.
  10. 10. Bottino R, Balamurugan AN, Smetanka C, et al. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies. Xenotransplantation, 2007, 14(1): 74-82.
  11. 11. Dufrane D, D’hoore W, Goebbels RM, et al. Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models. Xenotransplantation, 2006, 13(3): 204-214.
  12. 12. Rood PP, Buhler LH, Bottino R, et al. Pig-to-nonhuman primate islet xenotransplantation: a review of current problems. Cell Transplant, 2006, 15(2): 89-104.
  13. 13. Dufrane D, Goebbels RM, Fdilat I, et al. Impact of porcine islet size on cellular structure and engraftment after transplantation: adult versus young pigs. Pancreas, 2005, 30(2): 138-147.
  14. 14. Kim HI, Lee SY, Jin SM, et al. Parameters for successful pig islet isolation as determined using 68 specific-pathogen-free miniature pigs. Xenotransplantation, 2009, 16(1): 11-18.
  15. 15. Kim JH, Kim HI, Lee KW, et al. Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs. Xenotransplantation, 2007, 14(1): 60-66.
  16. 16. 蔣小峰, 錢(qián)滔來(lái), 向廣陽(yáng), 等. 五指山小型豬胰島分離和純化的初步研究. 臨床醫(yī)學(xué)工程, 2011, 18(9): 1343-1347.
  17. 17. Anazawa T, Balamurugan AN, Matsumoto S, et al. Rapid quantitative assessment of the pig pancreas biopsy predicts islet yield. Transplant Proc, 2010, 42(6): 2036-2039.
  18. 18. Jin SM, Shin JS, Kim KS, et al. Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation, 2011, 18(6): 369-379.
  19. 19. van der Windt DJ, Bottino R, Casu A, et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant, 2009, 9(12): 2716-2726.
  20. 20. d’Apice AJ, Cowan PJ. Gene-modified pigs. Xenotransplantation, 2008, 15(2): 87-90.
  21. 21. Ekser B, Cooper DK. Overcoming the barriers to xenotransplantation: prospects for the future. Expert Rev Clin Immunol, 2010, 6(2): 219-230.
  22. 22. Le Bas-Bernardet S, Tillou X, Poirier N, et al. Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons. Transplant Proc, 2011, 43(9): 3426-3430.
  23. 23. Goto M, Imura T, Inagaki A, et al. The impact of ischemic stress on the quality of isolated pancreatic islets. Transplant Proc, 2010, 42(6): 2040-2042.
  24. 24. 張明, 杜成友. 不同熱缺血時(shí)間對(duì)豬胰島獲取及功能的影響. 中國(guó)組織工程研究與臨床康復(fù), 2011, 15(5): 847-850.
  25. 25. Kuroda Y, Fujino Y, Morita A, et al. Oxygenation of the human pancreas during preservation by a two-layer (University of Wisconsin solution/perfluorochemical) cold-storage method. Transplantation, 1992, 54(3): 561-562.
  26. 26. Gioviale MC, Damiano G, Palumbo VD, et al. Pancreatic islets from non-heart-beating donor pig: two-layer preservation method in an in vitro porcine model. Int J Artif Organs, 2011, 34(6): 519-525.
  27. 27. Brandhorst D, Iken M, Brendel MD, et al. Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas. Transplant Proc, 2005, 37(1): 458-459.
  28. 28. Noguchi H, Ueda M, Nakai Y, et al. Modified two-layer preservation method (M-Kyoto/PFC) improves islet yields in islet isolation. Am J Transplant, 2006, 6(3): 496-504.
  29. 29. Brandhorst D, Iken M, Brendel MD, et al. Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation. Transplant Proc, 2005, 37(1): 229-230.
  30. 30. Taylor MJ, Baicu S, Greene E, et al. Islet isolation from juvenile porcine pancreas after 24-h hypothermic machine perfusion preservation. Cell Transplant, 2010, 19(5): 613-628.
  31. 31. 王玨, 侯宗柳, 李汝紅. 半自動(dòng)純化法分離純化成年豬胰島細(xì)胞的實(shí)驗(yàn)研究. 昆明醫(yī)學(xué)院學(xué)報(bào), 2011, 32(10): 14-18.
  32. 32. Yonekawa Y, Matsumoto S, Okitsu T, et al. Effective islet isolation method with extremely high islet yields from adult pigs. Cell Transplant, 2005, 14(10): 757-762.
  33. 33. Gray DW, Sudhakaran N, Titus TT, et al. Development of a novel digestion chamber for human and porcine islet isolation. Transplant Proc, 2004, 36(4): 1135-1138.
  34. 34. Kim SC, Han DJ, Kang CH, et al. Experimental islet isolation in porcine pancreas with new enzyme Liberase PI. Transplant Proc, 2004, 36(7): 2197-2199.
  35. 35. Bucher P, Bosco D, Mathe Z, et al. Optimization of neutral protease to collagenase activity ratio for islet of Langerhans isolation. Transplant Proc, 2004, 36(4): 1145-1146.
  36. 36. Brandhorst D, Huettler S, Alt A, et al. Adjustment of the ratio between collagenase class II and I improves islet isolation outcome. Transplant Proc, 2005, 37(8): 3450-3451.
  37. 37. Brandhorst H, Alt A, Huettler S, et al. The ratio between class II and class I collagenase determines the amount of neutral protease activity required for efficient islet release from the rat pancreas. Transplant Proc, 2005, 37(1): 215-216.
  38. 38. Hilling DE, Rijkelijkhuizen JK, Töns HA, et al. Porcine islet isolation outcome is not affected by the amount and distribution of collagen in the pancreas. Xenotransplantation, 2010, 17(3): 250-255.
  39. 39. Kinasiewicz A, Juszczak M, Pachecka J, et al. Pancreatic islets isolation using different protocols with in situ flushing and intraductal collagenase injection. Physiol Res, 2004, 53(3): 327-333.
  40. 40. Andrades P, Asiedu C, Ray P, et al. Islet yield after different methods of pancreatic liberase delivery. Transplant Proc, 2007, 39(1): 183-184.
  41. 41. Szot GL, Koudria P, Bluestone JA. Murine pancreatic islet isolation. J Vis Exp, 2007, (7): 255.
  42. 42. Atwater I, Guajardo M, Caviedes P, et al. Isolation of viable porcine islets by selective osmotic shock without enzymatic digestion. Transplant Proc, 2010, 42(1): 381-386.
  43. 43. 張偉杰, Schre J. 成年豬胰島的分離與純化. 中華器官移植雜志, 2000, 21(6): 341-343.
  44. 44. Qiao AY, Zhang WH, Chen XJ, et al. Isolation and purification of islet cells from adult pigs. Transplant Proc, 2010, 42(5): 1830-1834.
  45. 45. 徐艷艷, 傅紅興, 王浙明, 等. 大鼠胰島分離純化技術(shù)的改良及活性研究. 中國(guó)修復(fù)重建外科雜志, 2010, 24(4): 406-409.
  46. 46. 楊學(xué)偉, 章樂(lè)虹, 胡以則, 等. 成年豬胰島細(xì)胞的分離純化及其微囊包裹. 武漢大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2007, 28(7): 496-499.
  47. 47. 李鵬, 李汝紅, 趙維山, 等. 云南小耳豬胰島細(xì)胞的分離和純化方法. 昆明醫(yī)學(xué)院學(xué)報(bào), 2009, 30(12): 38-40.
  48. 48. Eckhardt T, Jahr H, Federlin K, et al. Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice. J Mol Med (Berl), 1999, 77(1): 123-125.
  49. 49. 張樺, 陳宏, 張振, 等. 碘克沙醇及Ficoll-400分離純化大鼠胰島細(xì)胞的效果對(duì)比. 世界華人消化雜志, 2007, 15(15): 1759-1762.
  50. 50. 黃躍南, 郭欣, 田素禮, 等. 成年豬胰島分離純化方法的改良. 世界華人消化雜志, 2005, 13(15): 1912-1914.
  51. 51. Min T, Yi L, Chao Z, et al. Superiority of visipaque (iodixanol)-controlled density gradient over Ficoll-400 in adult porcine islet purification. Transplant Proc, 2010, 42(5): 1825-1829.
  52. 52. 楊卿堯, 田郁文. 胰島細(xì)胞移植. 臺(tái)灣醫(yī)學(xué), 2007, 11(1): 42-46.
  53. 53. Shawl AI, Park KH, Kim UH. Insulin receptor signaling for the proliferation of pancreatic β-cells: involvement of Ca2+ second messengers, IP3, NAADP and cADPR. Islets, 2009, 1(3): 216-223.
  54. 54. Dekki N, Nilsson R, Norgren S, et al. Type 1 diabetic serum interferes with pancreatic beta-cell Ca2+-handling. Biosci Rep, 2007, 27(6): 321-326.
  55. 55. Corbin KL, Hall TE, Haile R, et al. A novel fluorescence imaging approach for comparative measurements of pancreatic islet function in vitro. Islets, 2011, 3(1): 14-20.
  56. 56. Elliott RB. Towards xenotransplantation of pig islets in the clinic. Curr Opin Organ Transplant, 2011, 16(2): 195-200.