| Objective: Study on how to improve the yield and quality of rat islets in isolation and purification by Ulinastatin. The effect of neovascu-larization by bFGF sustained release microencapsule was explored. Study on prevascularization subcutaneous transplantation of macroencapsulated SD rat islets normalized hyperglycemia in C57BL/6 diabetic mice. Whether prevascularization subcutaneous transplantation of SD rat islet coencap-sulated with bFGF, GF or NGF increased the survival rate of encapsulated SD rat islets was also studied.Methods: 1. Twenty male SD rats were randomly divided into 4 groups based on different methods of separation or purification methods of islets.①Group A :Islets were isolated by pancreatic ductal injection of collage-nase V and purified by a Ficoll gradient ,②Group B : Islets were isolated by pancreatic ductal injection of collagenase V and purified by transfer pipet hand-picked with optical microscopy ,③Group C : Islets were isolated by pancreatic ductal injection of collagenase V with Ulinastatin and purified by a Ficoll gradient,④Group D: Islets were isolated by pancreatic ductal injection of collagenase V with Ulinastatin and purified by transfer pipet hand-picked with optical microscopy .2, Twelve male C57BL/6 mice were randomly divided into 4 groups , Group A: 3μg bFGF sustained release microencapsule subcutaneous injection to mice, Group B: bFGF subcutaneous injection, Group C: subcutaneous injection of saline, Group D: subcutaneous injection of empty microencapsule. Subcutaneous angiogenesis was studied two weeks after injection.3, Fifteen subcutaneous prevascularized streptozotocin-induced diabetic C57BL/6 mice using alginate-encapsulated islets for rat-to-mice xenotransplantation were randomly divided into 3 groups, Group A: alginate-encapsulated rat islets for rat-to-mice prevascularization subcutaneous xenotransplantation, Group B: rat islets for rat-to-mice subcutaneous xenotransplantation, Group C: control. Blood glucose and body weight of STZ-induced diabetic mice after transplantation were observed every week. Histological study was performed on islets 8 weeeks after transplantation.4, Ten subcutaneous prevascularized STZ-induced diabetic C57BL/6 mices using microencapsulated islets for xenotransplantation were randomly divided into 2 groups, Group A: SD rat islets coencapsulated with bFGF for prevascularization subcutaneous transplantation, Group B: microcapsulated rat islets for prevascularization subcutaneous xenotransplantation. Blood glucose and body weight of STZ-induced diabetic mice after transplantation were observed. The amount of survival rat islets were counted 8 weeks after transplantation.5, Fifteen subcutaneous prevascularized STZ-induced diabetic mice using microencapsulated islets for transplantation were randomly divided into 3 groups, Group A: SD rat islet coencapsulated with GF for prevasculari zation subcutaneous transplantation, Group B: alginate-encapsulated rat islets for prevascularization subcutaneous transplantation, Group C: control. Blood glucose and body weight of STZ-induced diabetic mices after transplantation were observed. The amount of survival rat islets was counted 8 weeks after transplantation.6, Ten subcutaneous prevascularized STZ-induced diabetic C57BL/6 mice using encapsulated islets xenotransplantation were randomly divided into 2 groups, Group A: SD rat islet coencapsulated with NGF for prevascularization subcutaneous transplantation, Group B: encapsulated rat islets for prevascularization subcutaneous xenotransplantation. Blood glucose and body weight of STZ-induced diabetic mices after transplantation were observed. The amount of survival rat islets were counted 8 weeks after transplantation.Results: 1, The yield of purified islets was much more in group D than the other groups. The amount of islets which were isolated by pancreatic ductal injection of collagenase V with Ulinastatin were significantly more than that without Ulinastatin. The yield of islets purified by hand-picked was much more than that of purified by a Ficoll gradient.The characteristic of islets purified by Ficoll had complete outer membrane, smooth boundary and compact strcture, but small islet groups. The characteristic of islets purified by hand-picked had not only complete outer membrane, smooth boundary and compact strcture, but also more amount and more large islet groups. Purity of islet among the above groups were not significantly different(P>0.05).2, The amount of subcutaneous neovescular in group A is statistically significant more than that of group B, group C and group D (F=55.20, p<0.01).The amount of subcutaneous neovescular in group B were less than that of group A.3, Tail-vein blood glucose was measured after transplantation. The average blood glucose level of group A that received microencapsulated rat islets decreased 5 days after transplantation and achieved normo-glycemia 7 days posttransplantation. The xenografts functioned for 8 weeks for normoglycemia. The average blood glucose level of group B decreased , but did not received normoglycemia after transplantation, and started increasing from the third week, which was equal to that of an initial diabetic condition. The average blood glucose level of group C and group D sustained hyperglycemia , which is equal to that of an initial diabetic model.The body weight continuously increased for normal mice. However, the induction of diabetes by STZ injection resulted in the reduction of body weight. For the diabetic mice groups, the body weight continuously decreased after STZ injection and transplantation in group B, group C and group D. For group A, the microencapsulated islet xenotransplantation reversed the loss of body weight and led to a continuous increase in body weight.4, The average blood glucose level of group A and group B achieved normoglycemia 7 days posttransplantation. The xenotransplantation reversed the loss of body weight and led to a continuous increase in body weight, there was no considerable difference between theses two groups.Eight weeks after transplantation, the recipients were killed and subcutaneous implanted microencapsulated rat islets were retrieved, DTZ-positive microencapsules were counted. The group that islets coencapsulated with bFGF had more DTZ-positive microencapsules (t=8.22, P<0.01).5, The average blood glucose level of group A and group B achieved normoglycemia 7 days posttransplantation. The xenotransplantation reversed the loss of body weight and led to a continuous increase in body weight, there was no considerable difference between theses two groups.Eight weeks after transplantation, DTZ-positive microencapsules were counted. The group that islet coencapsulated with GF had more DTZ-positive microencapsules (F=36. 84, P<0.01).6, The average blood glucose level of group A and group B achieved normoglycemia 1 week posttransplantation. The xenotransplantation reversed the loss of body weight and led to a continuous increase in body weight, there was no considerable difference between theses two groups.Eight weeks after transplantation, DTZ-positive microencapsulated islets were counted. The group that islet coencapsulated with NGF had more DTZ-positive microencapsules(F=19. 84, P<0.01). Conclusion: 1, Ulinastatin perfusion could protect the islet from trypsin digestion in the process of rat separation, isolated by pancreatic ductal injection of collagenase V with Ulinastatin could receive more islets . The yield of islets purified by hand-picked was much more than that of purified by a Ficoll gradient. The characteristic of islets purified by hand-picked had not only complete outer membrane, smooth boundary and compact strcture, but also more amount and more large islet groups.2, Neovascularization was nessary for better and longer function of transplanted microencapsuled islets. bFGF sustained release microen-capsule was efficient and could induce effective angiogenesis .3, Prevascularization subcutaneous transplantation of encapsulated SD rat islet could normalize hyperglycemia in C57 diabetic mice. This approach had potential for favorable development of subcutaneous transplantation of microencapsulated islets as a cure for diabetes with minimally invasive surgery and repeated transplantation.4, Islet coencapsulated with bFGF for rat-to-mice prevascularization subcutaneous xenotransplantation was an effective approach for improved islet function after transplantation in a diabetic animal.5, Rat islets coencapsulated with GF prevascularization subcutaneous transplantation was an effective way for improved islet function and ameliorate effect after transplantation in a diabetic animal.6, Islets coencapsulated with NGF for xenotransplantation was an effective approach for improving islet function after transplantation in a diabetic animal. |
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