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Human proinsulin gene-transfected Chinese hamster ovary cells secrete immunoreactive insulin
Background:
Considering the difficulties in pancreas transplantation, the development of an artificial pancreas can be one of the new approaches. The present study was designed to assess whether or not Chinese hamster ovary (CHO) cells, which were transfected with the human proinsulin (hPI) gene, secrete immunoreactive insulin (IRI) and respond to glucose loading.
Materials And Methods:
A complementary DNA encoding hPI was obtained by polymerase chain reaction amplification from human pancreatic tissue and was inserted into the plasmid pcDNA I/NEO to construct an expression vector for the hPI gene. CHO cells were transfected with hPI gene using lipofectin, and the hPI gene-expressing clones (CHO/I) were selected.
Results:
Five clones of CHO/I cells, releasing IRI into the culture supernatant, were separated. Immunohistochemistry with a monoclonal antibody demonstrated the IRI in the cytoplasm of CHO/I cells, and transmission electron microscopic examination demonstrated the prominently developed mitochondria, but no secretion granules. ELISA assay demonstrated the secretion of IRI into the culture supernatant of CHO/I, but CHO/I cells did not respond to the glucose loading. When CHO/I cells were transplanted subcutaneously into the back of nude mice, the growing tumors secreted IRI.
Conclusions:
These results demonstrate that the hPI gene can be transfected into mammalian cells and function in vivo, and suggest that this kind of gene technology may be applicable in the development of an artificial pancreas.