Construction of an erythropoietin-expressing bioartificial renal tubule assist device

摘要

Methods: A eukaryotic expression vector pcDNA3.1-hEpo was constructed by standard cloning methods. G418 was applied to select for a stable erythropoietin (Epo) expression cell clone. Cell viability and cell growth curve were generated with MTT chromatometry. HK-2 cells were transfected with pcDNA3.1-hEpo, cultured, and then seeded into the hollow fibers of high-flux hemofilters. The expression levels of Epo in the culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). The inulin leak rate and the transport of Na and glucose (Glu) of the Epo-expressing bioartificial renal tubule assist device (RAD) were determined and compared with the conventional RAD. The specific inhibition of the transport by ouabain and phlorizin was determined. Results: The Epo expression plasmid pcDNA3.1-hEpo was successfully constructed and generated. Stable expression of Epo was obtained by selection of G418 after HK-2 cells were transfected. There was no obvious difference about cell growth curves between the transfected and untransfected HK-2 cells (p > 0.05). High levels of Epo expression were observed in both intra-and extraluminal culture media in the novel RAD. There was no significant difference about the inulin leak rate and the transport rate of Na and Glu between the novel and conventional RADs (p > 0.05 for each). And the transport function of the novel and conventional RADs was significantly inhibited by ouabain and phlorizin (p < 0.01 for each). Conclusions: A novel RAD that can express Epo was successfully constructed in vitro, in which other functions of the RAD were not affected by the transfection of pcDNA3.1-hEpo.