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- Title
Preliminary study on in-vitro trans-differentiation of non- β-cell into insulin-producing cells.
- Authors
Xia, H.; Zeng, S.; Lu, G.
- Abstract
Introduction: The limited availability of insulin-producing tissue hinders large-scale application of islet cell implantations for treating type I diabetic patients. The cell-replacement therapies for diabetics, as a treatment for diabetes, will become widely available only when new sources of pancreatic islets are found. Therefore, lots of researchers pay attention to transdifferentiation of non-β-cells into insulin-producing cells. Pancreatic and duodenal homeobox factor-1 (PDX-1) plays a central role in regulating pancreatic development and insulin gene transcription. 293T cell is a non-β-cell. In this study we transfected the plasmid of pAAV_MCS-PDX-1 into the 293T cell line to study if the genes related to pancreatic development are expressed in 293T cells. Materials/Methods: A 987-bp fragment of the PDX-1 cDNA was amplified by PCR from the human pancreas cDNA cloned into the EcoRI and XbaI sites of the pAAV_MCS plasmid. The 293T cells were cultured in DMEM (5.5 mM glucose) and transfected with the plasmid of pAAV_MCS-PDX- 1 and the plasmid of pAAV_MCS. 48 h later we extract the total RNA from 293T cells using TRIzol (Invitrogen). The purified RNA (2 µg) was reverse transcribed using Moloney murine leukaemia virus reverse transcriptase according to the manufacturer's instructions. PCR were performed with the appropriate oligonucleotide primers to analysis the expression of the genes relative to pancreatic development including PDX-1, ISL-1, PAX4, PAX6, Bate2, PC2 and INS. Results: At 48 h after transfection of the 293T with the plasmid of pAAV_MCS-PDX-1, RT-PCR analysis revealed that some genes relative to pancreatic development (PDX-1, ISL-1 and PAX6) were up-regulated; others (PAX4, Bate2 and PC2) were expressed whereas they are not expressed in untreated 293T cells. But the expression of the insulin gene has not been detected yet. Conclusions: The 293T cells are potential cells for being induced into insulin-producing cell lines.
- Subjects
PEOPLE with diabetes; ISLANDS of Langerhans
- Publication
Reproductive BioMedicine Online (Reproductive Healthcare Limited), 2008, Vol 16, Issue S2, pS-32
- ISSN
1472-6483
- Publication type
Abstract
- DOI
10.1016/S1472-6483(10)61544-1