【Abstract】ObjectiveTo develop a method of adult porcine pancreatic islet isolation.
MethodsThe tails of adult porcine pancreas were perfused through the pancreatic duct with 0.1% cold collagenase(type Ⅺ) and incubated at 38.5 ℃.The digested tissue was dispersed in 4 ℃ Hanks balanced salt solution(HBSS).The tissue suspensions were filtered through a 600 μm mesh.The residual tissue was resuspended in cold HBSS,and put in the Ricordi’s chamber and shaken for 5 minutes,then filtered again.The isolated islets were divided into three groups: control group(n=14),Pefabloc(trypsin inhibitor,n=8) group and FOY(trypsin inhibitor,n=5) group.The collagenase solution of the Pefabloc and FOY group was supplemented with 1.0 mmol/L Pefabloc and FOY respectively.
ResultsThe islet yields of the Pefabloc group and FOY group 〔(11 848±3 530) islet/g pancreas and (14 496±3 693) islet/g pancreas〕 were significantly higher than that of the control group 〔(8 505±3 349) islet/g pancreas〕,P＜0.05.The activity of pancreatic protein enzyme in digestive fluid after digestion in control group was higher than the activity of pancreatic duct before injection and Pefabloc group(P＜0.01),which the control group, pancreatic duct before injection and Pefabloc group were (114.7±50.0) BAEEU,(4.0±1.8) BAEEU and (5.5±2.7) BAEEU,respectively.The pancreatic duct before injection and Pefabloc group showed no significant difference in statistics. In control group,when the harvest of islet was more than 8 000/g,the activity of pancreatic protoin enzyme was less than that with the harvest of islet below 8 000/g 〔(78.3±26.7) BAEEU vs (137.5±48.4) BAEEU,P＜0.05〕.Islet after purification in control group,Pefabloc group and FOY group showed good insulin secretion ability for different concentration of glucose.
ConclusionA higher porcine pancreatic islet yield can be obtained by this method of pancreatic islet isolation and prophylactic administration of trypsin inhibitors consistently produce excellent islet yields.
Citation：
BAI Rixing,SONG Maomin.. Effect of Prophylactic Administration of Trypsin Inhibitors on Pancreatic Islet Isolation of Adult Porcine. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2005, 12(3): 275-279. doi:
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Ricordi C, Socci C, Davalli AM, et al. Isolation of the elusive pig islet [Ｊ]. Surgery, 1990; 107(6)∶ 688.
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Inoue K, Gu Y, Shinohara S, et al. Isolation of adult pig islet. In vitro assessment and xenotransplantation [Ｊ]. Int J Pancreatol, 1992; 12(2)∶173.
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Takei S, Teruya M, Grunewald A, et al. Isolation and function of human and pig islets [Ｊ]. Pancreas, 1994; 9(2)∶150.
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Muramatu M, Itoh T, Takei M，et al. Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase [Ｊ]. Biol Chem Hoppe Seyler, 1988; 369(7)∶617.
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Heiser A, Ulrichs K, MullerRuchholtz W. Influence of porcine strain, age, and pH of the isolation medium on porcine pancreatic islet isolation success [Ｊ]. Transplant Proc, 1994; 26(2)∶618.
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Socci C, Ricordi C, Davalli AM, et al. Selection of donors significantly improves pig islet isolation yield [Ｊ]. Horm Metab Res Suppl, 1990; 25(1)∶32.
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Ulrichs K, Bosse M, Wacker HH, et al. Histologic analysis of the porcine pancreas to improve islet yield and integrity after collagenase digestion [Ｊ]. Transplant Proc; 1994; 26(2)∶610.
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Sutherland DE, Steffes MW, Bauer GE, et al. Isolation of human and porcine islets of Langerhans and islet transplantation in pigs [Ｊ]. J Surg Res, 1974; 19(2)∶102.
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Heiser A, Ulriohs K, MullerRuchholtz W. Prophylactic trypsin inhibition during the isolation procedure guarantees reproducible islet yields [Ｊ]. Xenotransplantation,1994; 1(1)∶66.
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Toomey P, Chadwick DR, Contractor H, et al. Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion [Ｊ]. Br J Surg, 1993; 80(2)∶240.
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Buhler L, Deng S, Mage R, et al. Pig islet isolation: new aspects [Ｊ]. Transplant Proc, 1994; 26(2)∶628.
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张伟杰，Scherzen MJ. 成年猪胰岛分离与纯化 [Ｊ]. 中华器官移植杂志， 2000； 21（11）∶341.
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孙铭，宁刚，李涛，等. 微囊杂种猪胰岛移植治疗 Ⅰ 型糖尿病的实验研究 [Ｊ]. 中国生物医学工程学报， 2002； 21（4）∶102.
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- 1. Ricordi C, Socci C, Davalli AM, et al. Isolation of the elusive pig islet [Ｊ]. Surgery, 1990; 107(6)∶ 688.
- 2. Inoue K, Gu Y, Shinohara S, et al. Isolation of adult pig islet. In vitro assessment and xenotransplantation [Ｊ]. Int J Pancreatol, 1992; 12(2)∶173.
- 3. Takei S, Teruya M, Grunewald A, et al. Isolation and function of human and pig islets [Ｊ]. Pancreas, 1994; 9(2)∶150.
- 4. Muramatu M, Itoh T, Takei M，et al. Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase [Ｊ]. Biol Chem Hoppe Seyler, 1988; 369(7)∶617.
- 5. Heiser A, Ulrichs K, MullerRuchholtz W. Influence of porcine strain, age, and pH of the isolation medium on porcine pancreatic islet isolation success [Ｊ]. Transplant Proc, 1994; 26(2)∶618.
- 6. Socci C, Ricordi C, Davalli AM, et al. Selection of donors significantly improves pig islet isolation yield [Ｊ]. Horm Metab Res Suppl, 1990; 25(1)∶32.
- 7. Ulrichs K, Bosse M, Wacker HH, et al. Histologic analysis of the porcine pancreas to improve islet yield and integrity after collagenase digestion [Ｊ]. Transplant Proc; 1994; 26(2)∶610.
- 8. Sutherland DE, Steffes MW, Bauer GE, et al. Isolation of human and porcine islets of Langerhans and islet transplantation in pigs [Ｊ]. J Surg Res, 1974; 19(2)∶102.
- 9. Heiser A, Ulriohs K, MullerRuchholtz W. Prophylactic trypsin inhibition during the isolation procedure guarantees reproducible islet yields [Ｊ]. Xenotransplantation,1994; 1(1)∶66.
- 10. Toomey P, Chadwick DR, Contractor H, et al. Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion [Ｊ]. Br J Surg, 1993; 80(2)∶240.
- 11. Buhler L, Deng S, Mage R, et al. Pig islet isolation: new aspects [Ｊ]. Transplant Proc, 1994; 26(2)∶628.
- 12. 张伟杰，Scherzen MJ. 成年猪胰岛分离与纯化 [Ｊ]. 中华器官移植杂志， 2000； 21（11）∶341.
- 13. 孙铭，宁刚，李涛，等. 微囊杂种猪胰岛移植治疗 Ⅰ 型糖尿病的实验研究 [Ｊ]. 中国生物医学工程学报， 2002； 21（4）∶102.
