Diabetologia · 2006
A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Barbu A R, Bodin B, Welsh M, Jansson L, Welsh N
Аннотация
AIMS/HYPOTHESIS: Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets.
MATERIALS AND METHODS: We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion.
RESULTS: In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure.
CONCLUSIONS/INTERPRETATION: These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.
MATERIALS AND METHODS: We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion.
RESULTS: In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure.
CONCLUSIONS/INTERPRETATION: These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.