Reversible Immortalization Of Rat Pancreatic β Cells Mediated By Cre/LoxP Site-specific Recombination With SV40LTAg Alone Or SV40LTAg,hTERT Together

Background and objective:Type 1 diabetic patients are insulin dependent as a result of autoimmune destruction of pancreaticβcells. It is now well established that in later stages of type 2 diabetes,β-cell mass is reduced by about 50%, and most of these patients require exogenous insulin to control hyperglycemia. Exogenous insulin administration cannot ensure continuous blood glucose control and satisfactory prevention of diabetes complications. Recent studies have demonstrated that islet allograft transplantation can be a successful therapeutic option in the treatment of patients with Type I and Type II diabetes. However, this impressive recent advance is accompanied by a very important constraint. There is a critical paucity of pancreatic islets or pancreaticβcells for islet transplantation to become a large-scale therapeutic option in the field. Expansion of primaryβcells by growth factors is also limited and hampered by the senescence of the cells. Reversible immortalization with Cre/LoxP site-specific recombination and transgenic technology can be potentially a new way to solve this problem. At present, reversible immortalized hepatic cell, fibroblast, phaeochrome cell and neuron stem cell have been successfully established by simian virus 40 large T antigen gene(SV40LTAg)or human telomerase reverse transcriptase(hTERT). However, some researchers have found that transduction of the hTERT gene and induction of telomerase activity have no observable effect on the proliferative life span of pancreatic islet cell cultures. Notably, both telomere-dependent and telomere-independent models of senescence have implicated p16^INK4A and p21^CIP1/WAF1. And SV40LTAg interferes with both p16/Rb and p53/p21 pathways. Therefore, we are going to transduce SV40LTAg into primary rat pancreaticβcells to establish immortalizedβcells. Although hTERT alone could not induceβcells to immortalization, is there any difference betweenβcells transduced with SV40LTAg alone or SV40LTAg,hTERT together? In order to explore a convenient and pragmatic way to obtain availableβcells with expansion ability, we are going to transduce SV40LTAg alone or SV40LTAg, hTERT together to establish two kinds of immortalizedβcells, after their continuous proliferation in vitro, then excise the immortalizing genes with Cre/LoxP system to obtain two kinds of revertedβcells respectively, and investigate their phenotype, insulin producing function and tumorigenesis.Methods:(1) The reversible immortalization retroviral vector phTERT-I-GTKlox containing hTERT gene was constructed. The reversible immortalization retroviral vector pLCRSTP containing SV40LTAg and Cre-ER gene was identified.(2) Primary rat pancreatic islets were isolated by collagenase infusion digestion in the choledochous duct and purified with Ficoll400 gradient centrifugation. The islets'purity and motility were evaluated by DTZ staining, AO-PI staining and immunofluorescence staining targeting insulin and Pdx-1.(3) pLCRSTP and phTERT-I-GTKlox retrovirus particles were packaged by the Phoenix Amphotropic packaging cell line.(4) The primary rat pancreatic islet cells were infected by pLCRSTP retrovirus particles, and the immortalizedβcells , named LT-β, were obtained through the limiting dilution culture. LT-βcells were infected by phTERT-I-GTKlox retrovirus particles, and the immortalizedβcells , named LT-hTERT-β, were also obtained through the limiting dilution culture.(5) Tamoxifen treatment induced the translocation of Cre-ER protein from the cytoplasm to the nucleus. The immortalizing genes and Cre-ER gene itself were excised in both of the LT-β(P20) and LT-hTERT-β(P20) cells. After ganciclovir treatment, the reverted cells, respectively named R-LT-βand R-LT-hTER, were both obtained.(6) The integration, expression and intracellular location of SV40LTAg,Cre-ER and hTERT genes were analyzed by polymerase chain reaction(PCR), reverse transcription polymerase chain reaction(RT-PCR), western blot and immunocytochemistry techniques in LT-β(P20),LT-hTERT-β(P20) cells. The excision of these genes were analyzed by the same techniques in R-LT-β,R-LT-hTERT-βcells. EGFP expression in LT-hTERT-β(P20) and R-LT-hTERT-βcells were observed under the laser confocal microscopy. (7) The cell growth rates were assessed by MTT colorimetric assay. The glucose- stimulated insulin secretion were determined. The mRNA expression of Pdx-1, somatostatin and glucagon were analyzed by semi-quantity RT-PCR. The expression and intracellular location of insulin and Pdx-1 were analyzed by immunocytochemistry staining. The tumorigenesis were evaluated by chromosome karyotype analysis, plate clone formation tests and transplantation tests in SCID mice.Results:(1) The reversible immortalization retroviral vector phTERT-I-GTKlox containing hTERT gene was successfully constructed. The reversible immortalization retroviral vector pLCRSTP containing SV40LTAg and Cre-ER gene was successfully identified.(2) Primary islet cells with high purity and motility were successfully isolated, purified, cultured and identified.(3) pLCRSTP-Phoenix A cells stably packaging pLCRSTP retrovirus particles were successfully obtained.(4) Two kinds of immortalizedβcells, LT-βand LT-hTERT-β, were successfully obtained.(5) Two kinds of revertedβcells, R-LT-βand R-LT-hTERT-β, were successfully obtained after the excision of SV40LTAg,Cre-ER and hTERT genes by Cre-ER/LoxP system.(6) The integration, expression and correct intracellular location of SV40LTAg,Cre-ER and hTERT genes were identified in LT-β(P20),LT-hTERT-β(P20) cells. The excision of these genes were identified in R-LT-β,R-LT-hTERT-βcells. EGFP expression was identified in LT-hTERT-β(P20) and R-LT-hTERT-βcells.(7) LT-β,LT-hTERT-βcells were always in their exponential phase of growth. Their doubling generation time were shorter than that of primary islet cells(P <0.01), and there was no difference between LT-βand LT-hTERT-βcells. R-LT-βand R-LT-hTERT-βcells'expansion were extremely limited, with much longer doubling generation time compared with immortalizedβcells(P <0.01), and consistent with that of primary islet cells.(8) In response to low glucose or high glucose, both of the secretion of insulin from the LT-β(P20) and LT-hTERT-β(P20) cells were low. Both of the secretion of insulin from the R-LT-βand R-LT-hTERT-βcells were higher than those of the immortalizedβcells(P <0.01), and consistent with that of primary islet cells. Both of the insulin-releasing index in the LT-β(P20) and LT-hTERT-β(P20) cells were low, Both of the insulin-releasing index in the R-LT-βand R-LT-hTERT-βcells were higher than those of the immortalizedβcells (respectively P <0.01 & P <0.05), and consistent with that of primary islet cells. Between the two kinds of immortalized cells and between the two kinds of reverted cells, there was no difference on the insulin secretion as well as insulin-releasing index.(9) The Pdx-1 mRNA were more strongly expressed in R-LT-βand R-LT-hTERT-βcells than in LT-β(P20) and LT-hTERT-β(P20) cells(P <0.01), although less than primary islet cells, respectively 0.89 fold & 0.91 fold of that in primary islet cells. Between the two kinds of immortalized cells and between the two kinds of reverted cells, there was no difference on the Pdx-1 mRNA expression.(10) There was no evidence of somatostatin or glucagon mRNA expression in any of the LT-β(P20), LT-hTERT-β(P20), R-LT-βand R-LT-hTERT-βcells.(11) The expression and correct intrarcellular location of insulin and Pdx-1 protein were identified in both of the two revertedβcells, and consistent with that in primary islet cells.(12) Chromosome karyotype analysis revealed a normal number of chromosome. Plate clone formation tests and transplantation tests in SCID mice revealed no evidence of tumorigenesis.Conclusions:(1) The reversibly immortalizedβcell LT-βis successfully obtained by"one-step"transfection of pLCRSTP. The reversibly immortalizedβcell LT-hTERT-βis successfully obtained by"two-step"transfection of pLCRSTP and phTERT-I-GTKlox. Both of them can continuously proliferate in vitro with long-term stable passage.(2) Two kinds of revertedβcells, R-LT-βand R-LT-hTERT-β, are successfully obtained after the excision of SV40LTAg,Cre-ER and hTERT genes by Cre-ER/LoxP system.(3) Both of the secretion of insulin from the R-LT-βand R-LT-hTERT-βcells are higher than those of the immortalizedβcells, and consistent with primary islet cells. The Pdx-1 mRNA are more strongly expressed in R-LT-βand R-LT-hTERT-βcells than in LT-β(P20) and LT-hTERT-β(P20) cells, although less than primary islet cells, respectively 0.89 fold & 0.91 fold of that in primary islet cells. There is no evidence of somatostatin or glucagon mRNA expression in any of the LT-β(P20), LT-hTERT-β(P20), R-LT-βand R-LT-hTERT-βcells. Chromosome karyotype analysis, plate clone formation tests and transplantation tests in SCID mice reveals no evidence of tumorigenesis. Both of R-LT-β, R-LT-hTERT-βcells possess satisfactory phenotype and insulin producing function.(4) Between the two kinds of immortalizedβcells and between the two kinds of revertedβcells, there is no evidence of difference on the cell growth rates, the secretion of insulin in response to low glucose or high glucose, the insulin-releasing index and the mRNA expression of Pdx-1.