[Differentiation of polygene-modified bone marrow mesenchymal stem cells into insulin-producing cells].

OBJECTIVE

To evaluate the effects of insulin gene transcription regulators PDX-1, NeuroD1 and MafA on the differentiation of bone marrow mesenchymal stem cells (mMSCs) into insulin-producing cells.

METHODS

Murine mMSCs were isolated, cultured and expanded. The base sequences of transcription factors PDX-1, NeuroD1 and MafA were obtained by total gene synthesis and the recombinant adenovirus vectors harboring target genes constructed and transfected into packaging cell line 293A. mMSCs were infected with adenovirus separately or together, and then differentiated in vitro into insulin-producing cells. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to detect insulin gene expression, immunofluorescence for identifying the presence of insulin protein and insulin enzyme-linked immunosorbent assay (ELISA) for evaluating the secretory volume of insulin.

RESULTS

The differentiation extent of mMSCs into β-cell was analyzed. The β-cell-specific transcriptional regulators and insulin gene were expressed in mMSCs after transfection. Immunofluorescent analyses revealed the activated expression of insulin in the cytoplasm of differentiated cells. A significant content of insulin was released in these cells in response to a certain concentrations of glucose stimulation. The insulin content of mMSCs infected with a combination of three transcription factors was significantly higher than that of the control group [(112.84 ± 9.67) mU/L vs (1.60 ± 0.22) mU/L, P < 0.05].

CONCLUSION

After modification by transcriptional factors PDX-1, NeuroD1 and MafA, mMSCs can secrete insulin through starting endogenous insulin gene transcription.