Construction Of Recombinant Retroviral Vector Containing Glucose Transporter-2 Gene (GLUT-2) And Functional Study Of INS-1 Cell Stable Transfected By GLUT-2

Aim:In the patients with chronic pancreatitis, the insulin secretion is significantly reduced due toβ-cell damage. While long term high glucose toxicity could accelerateβcell apoptosis and cause secondary diabetes. Therefore, control blood glucose level is a key factor in the treatment of chronic pancreatitis. Unfortunately, there are many drawbacks in the current treatment. Traditional insulin injection is prone to causing complications by blood sugar fluctuations.Recently pancreatic islet cell transplantation has been considered as an ideal therapeutic approach; however the separation and storage ofβcells bring a new obstacle. Thus, looking for viable new sources forβcell transplantation becomes a research focus.In this project, human glucose transporter-2 (GLUT-2) gene mediated by a retrovirus vector was transfected into INS-1 cell line in order to simulate the insulin secretion under physiologic conditions. And this "artificial beta cell" can provide a new and easy source of transplantation.Backgrounds:INS-1 cell line with an insulin content of 8 ug/106 cells was established by dispersion of a radiation-induced insulinoma from NEDH rats. The insulin secretion of INS-1 is similar to the primaryβcells. Furthermore, it could not secrete glucagon and somatostatin which rules out other effects on the study of insulin secretion.1. Insulin release is in proportion to the glucose metabolic rate ofβcells. Therefore, the proteins or enzymes which are able to regulate the amount of glucose inβcells were serves as glucose sensors. glucose transporter 2 ( GLUT-2) is one of the glucose sensors. The activity GLUT-2 is modulated by the level of blood sugar which enables it to regulate the glucose metabolic rate inβcells.2. retroviral vector could stably express the target gene in host cells which is the most commonly used system in the study of "artificial beta cell".Part I: GSIS Assay for INS-1 cellsObjective: to detect the glucose-stimulated insulin secretion (GSIS) level in cultured INS-1 cells.Method: (1) INS-1 cell line was cultured in RPMI-1640 containing 10 % of fetal bovine serum (FBS) at 37 C. (2) GSIS assayResult: (1) INS-1 cells grew well in RPMI-1640 containing 10 % of FBS. (2) There is insulin secretion in cultured INS-1 cells according to GSIS assay, however there is significant different of GSIS between cultured cell line and physiologic conditions.Conclusion: we successfully cultured INS-1 cell line. There is insulin secretion in INS-1 cell line, but it is significant different with the physiologic conditions.Part II Construction of Recombinant Retroviral Vector containing GLUT-2 geneObjective: In order to transfect INS-1 cells with GLUT-2 gene, Recombinant Retroviral Vector containing GLUT-2 gene (pL-GLUT2-SN) was constructed and packed with PA317 cells. And the clones producing high titer virus were selected.Method: Plasmid pcB7/GLUT2 was cloned into Retroviral Vector pLXSN to construct Recombinant Retroviral Vector pL-GLUT2-SN. pL-GLUT2-SN was identified by sequencing and restriction enzyme digestion. The pL-GLUT2-SN was transfected into PA317 cells using Lipofectamine 2000. The integration and transcription of GLUT-2 gene in PA317 cells were detected by PCR and RT-PCR.Result: (1)the recombinant vector pL-GLUT2-SN was double digested by EcoRI and BamHI to obtain a 1996 bp target band. And the sequencing result of GLUT-2 cDNA indicates that we obtained the right vector. Then the G418 resistant clone PA317/GLUT2 was obtained, and the highest titer is 7.1×10~5 CFU/mL. (2)There was a 158 bp fragment in the RT-PCR result of PA317/GLUT2, which demonstrated that GLUT-2 was expressed in PA317/ GLUT-2 cell.Conclusions: PA317/GLUT2 cells were constructed, high titer virus was determined and the integration and transcription of GLUT-2 were identified.Part III Construction of INS-1 cell stably expressing GLUT-2 gene and GSIS assayObjective: Construct the stably expression GLUT-2 gene INS-1 cell line and perform GSIS assay to lay a strong foundation of in vivo experiment.Method: (1)INS-1 cells were infected by GLUT2 virus. And the expression of GLUT-2 in INS-1/GLUT-2 cells was identified by RT-PCT and western blot. (2) GSIS assay was performed with INS-1/GLUT-2 cells.Result: (1) There is higher GLUT-2 expression in INS-1/GLUT-2 cells than INS-1 cells by RT-PCR. (2) Western blot indicated that GLUT-2 was over expressed in INS-1/GLUT-2 cells. (3) GSIS curve of INS-1/GLUT-2 cells was shift to the right compared with INS-1 cell line.Conclusion: We constructed a novel "artificial beta cell" INS-1/GLUT-2 by transfecting INS-1 cells with retroviral vector mediated GLUT-2 gene. The insulin secretion of INS-1/GLUT-2 cells can mimic the physiologic conditions which provide a new source for pancreatic islet cell transplantation.