AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intr...AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase diges- tion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad- HO-1) or enhanced green fluorescent protein gene (Ad- EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmuno- assay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% ± 6% vs 52% ± 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 ± 0.55 mIU/ L/30IEQ vs 4.57 ± 0.40 mIU/L/30IEQ, 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets(71% ± 15% vs 52% ± 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 ± 2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ; 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P < 0.05). CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.展开更多
Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic ...Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic recipient;the intrahepatic site may not be the most appropriate site for islet implantation;and immunosuppressive regimens,which are associated with side effects,are often required to prolong survival of the islet graft.An alternative source of insulinproducing cells would therefore be of major interest.Pigs represent a possible alternative source of beta cells.Grafting of pig islets may appear difficult because of the immunologic species barrier,but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression.Therefore,a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation.Although several groups have shown that encapsulated pig islets are functional in small-animal models,less is known about the use of bioartificial pancreases in large-animal models.In this review,we summarize current knowledge of encapsulated pig islets,to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.展开更多
Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombin...Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombinant adenovirus vector containing human HO-1 gene（Ad-HO-1 ） or enhanced green fluorescent protein gene（Ad-EGFP） was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index（SI） was calculated. Glucose-stimulated insulin release was used ＇to assess islet viability. Results：Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index （SI） of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets （P 〈 0.05）. Conclusion： Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.展开更多
Objective： To observe the effect of glucagon-like peptide 1 （GLP-1） on the gene expressions of transcription factors （PDX-1, PAX-6 and NKx2.2 ） in freshly isolated rat pancreatic islets and investigate the associ...Objective： To observe the effect of glucagon-like peptide 1 （GLP-1） on the gene expressions of transcription factors （PDX-1, PAX-6 and NKx2.2 ） in freshly isolated rat pancreatic islets and investigate the associated physiological and therapeutic implication of GLP-1. Methods： The isolated rat islets were incubated with 10 nmol/L GLP-1 for 1, 3 and 5 days, respectively. Total cellular RNA was extracted and the expressions of PDX-1, PAX-6 and NKx2.2 gene were detected by semiquantity RT-PCR. Results： Compared with the control group, the PDX-1, PAX-6 and NKx2.2 gene expressions were significantly increased after co-cultured with GLP-1 for 1 day （P 〈 0.05）. The effect was shown in a time-dependent manner. All three gene expressions reached the peak on the 5th day. Conclusion： GLP-1 can improve the function of pancreatic islet by regulating the gene expressions of transcription factors in β cells.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30571759Social Development Foundation of Shanghai, No. 200253
文摘AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase diges- tion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad- HO-1) or enhanced green fluorescent protein gene (Ad- EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmuno- assay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% ± 6% vs 52% ± 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 ± 0.55 mIU/ L/30IEQ vs 4.57 ± 0.40 mIU/L/30IEQ, 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets(71% ± 15% vs 52% ± 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 ± 2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ; 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P < 0.05). CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.
基金Supported by European Grant Titled Xenome,UE LSHBCT-2006-037377
文摘Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic recipient;the intrahepatic site may not be the most appropriate site for islet implantation;and immunosuppressive regimens,which are associated with side effects,are often required to prolong survival of the islet graft.An alternative source of insulinproducing cells would therefore be of major interest.Pigs represent a possible alternative source of beta cells.Grafting of pig islets may appear difficult because of the immunologic species barrier,but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression.Therefore,a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation.Although several groups have shown that encapsulated pig islets are functional in small-animal models,less is known about the use of bioartificial pancreases in large-animal models.In this review,we summarize current knowledge of encapsulated pig islets,to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.
基金This work was supported by National Natural Science Foundation of China, No 30571759
文摘Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombinant adenovirus vector containing human HO-1 gene（Ad-HO-1 ） or enhanced green fluorescent protein gene（Ad-EGFP） was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index（SI） was calculated. Glucose-stimulated insulin release was used ＇to assess islet viability. Results：Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index （SI） of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets （P 〈 0.05）. Conclusion： Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.
基金135 Project Foundation of Jiangsu Province (NO2001)
文摘Objective： To observe the effect of glucagon-like peptide 1 （GLP-1） on the gene expressions of transcription factors （PDX-1, PAX-6 and NKx2.2 ） in freshly isolated rat pancreatic islets and investigate the associated physiological and therapeutic implication of GLP-1. Methods： The isolated rat islets were incubated with 10 nmol/L GLP-1 for 1, 3 and 5 days, respectively. Total cellular RNA was extracted and the expressions of PDX-1, PAX-6 and NKx2.2 gene were detected by semiquantity RT-PCR. Results： Compared with the control group, the PDX-1, PAX-6 and NKx2.2 gene expressions were significantly increased after co-cultured with GLP-1 for 1 day （P 〈 0.05）. The effect was shown in a time-dependent manner. All three gene expressions reached the peak on the 5th day. Conclusion： GLP-1 can improve the function of pancreatic islet by regulating the gene expressions of transcription factors in β cells.
