Experimental study of adipose-derived mesenchymal stem cells in rats transfected into the insulin-secreting cells in vitro

Abstract

Objective To investigate the possibility of transfection of adipose-derived mesenchymal stem cells (ADMSCs) into the insulin-secreting cells in vitro,and assay the secretion of insulin of ADMSCs in high and low glucose environment.Methods The ADMSCs that untransfected were in the control group,the ADMSCs that contained PcDNA3.1 were in the vacant vector group,and the ADMSCs that contained PcDNA3.1-hINS were in the recombinant vector group.After transfection,the recombinant vector group were sub-divided into the 1,6,12,18 days groups.According to the concentrations of glucose,the recombinant vector 18 days group were divided into the high glucose group and low glucose group.Human insulin gene was amplified by RT-PCR,and the eukaryotic expression recombinant vector PcDNA3.1-hINS that contained the human insulin gene was constructed.The ADMSCs were obtained by digestion and centrifugation,and then underwent flow cytometry for identification.The transcription of insulin DNA was assayed by RT-PCR,and the levels of insulin were assayed by ELISA.Glucose test was done in the recombinant vector 18 days group.The measurement data was shown in the format of (x) ± s,the measurement data in multiple groups were compared by randomized analysis of variance,and the comparison among groups was performed by the t test.ResuIts The expressions of CD44,CD90,CD106 were positive,and the expressions of CD34,CD45 and CD11b were negative.No insulin DNA transcription was detected in the control group and vacant vector group.The levels of insulin secreted were (4.7 ± 0.8) mIU/L,(8.8 ± 0.5) mIU/L,(8.9 ± 0.8)mIU/L,(8.6 ± 0.6)mIU/L in the recombinant vector 1,6,12,18 days group,which were significantly higher than (1.3 ± 0.6) mIU/L in the control group and (1.7 ± 0.8) mIU/L in the vacant vector group (t =10.09,32.64,22.20,55.53 ; 9.23,27.56,19.43,51.25,P ＜ 0.05).There were significant differences in the levels of insulin secreted between the recombinant vector 1 day group and the recombinant vector 6,12,18 days groups (t =12.77,12.26,13.93,P ＜0.05).There were no significant difference in the levels of insulin secreted between the recombinant vector 6,12,18 days groups (F =45.67,P ＞ 0.05).There was a significant difference in the level of insulin secreted between the high glucose group and the low glucose group (t =2.03,P ＜ 0.05).The result of the glucose stimulation test was negative.Conclusion The ADMSCs are transfected into insulinsecreting cells in vitro successfully,and the secretion of insulin is stable.Although the secretion of insulin can't change in line with the concentration of glucose,it is a new seed cell for the treatment of diabetes with stem cells. Key words: Dipose-derived mesenchymal stem cells; Diabetes; Transfection; Stem cells