| Objective By establishing reversible immortalized murine pancreaticβcells through SV40T regulated by Tet-on advanced system, we can observe the biological characteristic of immortalized pancreatic (3 cells, and evaluate the functional and security of the reversible immortalization of mouse pancreaticβcells. The results of this study will provide experimental date for the clinic pancreatic cell transplant therapy.Methods Using Gateway recombination technology, the lentiviral vectors pLV.EX2d.P/puro-EF1A> rtTA (M2) and pLV.EX3d.P/neo-TRE> SV40T> IRES/eGFP that under Tet-on advanced system control were constructed. Then both of them were transfected into packaging cells 293FT by lipofectin-2000 transfection, to obtain high titer of virus particles. The primary pancreatic islet cells of mouse were infected by the lentiviral virus particles,24 hours after infected, doxycycline was added to induced the expression of SV40T, And 48 hours after infected puromycin and G418 were added to obtain the selected immortalizedβcells. Then through ELISA tests and RT-PCR, the detection of basic insulin secretion and pancreaticβcell related genes were detected, and the reversible immortalization of mouse pancreaticβ-cells with physiological function were obtained, named RIB cells. Observed changes in biological characteristics of RIB cells under the induction of DOX the expression of SV40T and P-cell related genes in mRNA and protein levels was detected by RT-PCR and immunefluorescence; and some method including karyotype analysis, plate colony formation assay, cell growth ability and detective glucose-stimulated insulin release test, were employed to detected the function and security of RIB cells that with and without DOX regulation.Results RT-PCR result showed that recombinant lentiviral vector pLV.EX2d.P/puro-EF1A> rtTA (M2) and pLV.EX3d.P/neo-TRE> SV40T> IRES/eGFP was constructed and validation; Transfected into 293 cells can obtain high titers of virus particles; infected the primary mouse pancreatic islet, successfully establishs a reversible immortalized cells that named RIB cells which can express Pdxl, Nkx6.1, Glut-2, Pax6, Insulin. The expression of SV40T of RIB cells in mRNA and protein levels; RIB cells had strong proliferation capacity, the expression of SV40T was stopped when DOX was absenced, and the reverted RIB cell hold the decreased proliferation rate and stoped proliferation in 5 days; P cell-related genes and proteins expression of RIB cells in whole process both the DOX presence or absence; Chromosome karyotype analysis revealed a normal number of chromosome; Plate clone formation tests showed no tumorigenesis. Reverted RIB cell loss the proliferation of immortalized after removal DOX. In response to glucose stimulation test, the insulin secretion of RIB cells was weak; however the insulin secretion of Reverted RIB cell was increased, but can reach 1/3-1/2 of normal islet secretion amount.Conclusion The study successfully constructed lentiviral vector pLV.EX2d.P/puro-EF1A> rtTA (M2) and pLV.EX3d.P/neo-TRE> SV40T> IRES/eGFP,and established RIB cell line. The characteristics of RIB cell were with long term stable survival period, continuously proliferate in vitro, normal karyotyotype, and expression of pancreatic cell related genes and protein. The reverted RIB cell stop proliferate and did not express the SV40T gene. And the Insulin secretion capacity of reverted RIB cell is stronger than RIB, but still not bestill is not compared withas good as primary islet cells. |
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