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Abstract
Background
A major obstacle of islet transplantation is an inadequate supply of insulin-producing tissue. Ad-PDX-1/VP16 overexpression and Exendin-4 treatment have been proved the effects on differentiation and proliferation of pancreatic stem cells. But, the study is insufficient using adult animal pancreatic stem cells.
Methods
Pancreatic cells were prepared from the non-endocrine fraction of canine pancreases. This cells were cultivated free floating state and monolayer culture after dispersion. The floating pancreatic cells were transplanted under the kidney capsule of normoglycaemic nude mice. The dispersed pancreatic cells were infected with Ad-PDX-1/VP16 or Ad-GFP. After infection, those cells were transplanted of nude mice. After transplantation, mice were treated with either 1 nmol/kg exendin-4 or saline solution by intraperitoneal injection for 10 days.
Results
The relative volume of the beta-cells in the grafts of the free floating cultured pancreatic cells were 23.4 ± 13.1% at two weeks and 5.2 ± 2.0% at eight weeks. At two weeks after transplantation, the relative volume of insulin-positive cells in the grafts of dispersed pancreatic cells were 28 ± 5.7%, 20.5 ± 0.7% and 31 ± 1.4% in control, GFP and PDX-1/VP16 treated groups respectively. At eight weeks after transplantation, the relative volume of insulin-positive cells in the grafts were 11.8 ± 5.9%, 8 ± 7.3% and 16.6 ± 7.4% in control, GFP and PDX-1/VP16 treated groups respectively. Exendin-4 treatment didn't show any additive effects on transdifferentiation of pancreas stem cell into beta-cells.
Conclusion
The expansion and transdifferentiation were not observed after the transplantation of the free floating cultured pancreatic cells. PDX-1/VP16 overexpression induces the transdifferentiation of adult pancreatic cells into beta-cells. However Exendin-4 treatment hasn't any effects on the expansion and transdifferentiation of the cells in the grafts.
Figures and Tables
Fig. 1
Schematic of experimental design.
A, General scheme of pancreatic cell culture system were separated into two groups: floating and monolayer culture. In monolayer culture, we used an Adenovirus for PDX-1 expression. Ad-GFP was used as an adenoviral control; B, Schematic diagram of experimented design. On day 0, Ad-GFP or Ad-PDX-1/VP16 infected pancreatic cell were transplanted under the kidney capsule of normoglycaemic nude mice. After 1 day, nude mice was treated with a daily injection of Exendin-4 (1 nmol/kg, 100 µL) or saline (0.9% NaCl, 100 µL) for 10 days. Finally, all grafts were harvested on the 2 weeks and 8 weeks.
Fig. 2
Adenovirus-mediated expression of GFP and PDX-1 in the pancreatic cell.
Ad-GFP (A, top) and Ad-PDX-1/VP16 (A, bottom) were visible 48 hours after infection in about 80% and 70%, respectively. (B) Flow cytometric analysis of virus expression. GFP+ cells were isolated in adenovirus-infected disperse pancreatic cells.
Fig. 3
Effect of exendin-4 on beta-cell specific gene expression in canine pancreatic cells. PDX-1 and insulin were estimated by RT-PCR. The bar graphs show quantification of the results of real-time PCR. The mRNA levels of these genes were normalized to GAPDH (B, C). Results are expressed as means ± S.E for n = 3 independent experiment.
*P ≤ 0.05.
Fig. 4
Insulin immunohistochemical staining in pancreatic cell graft. (A) The canine pancreatic cells were stained with insulin immunohistochemical staining at 0 day. The canine pancreatic cells of graft was stained with insulin immunohistochemical staining at 2 week (B) and 8 weeks (C). (D) Beta-cell percentage in transplanted pancreatic cell of graft. (Original magnification ×400)
*P ≤ 0.05.
**P ≤ 0.001.
Fig. 5
Insulin immunohistochemical staining in pancreatic cell graft and the effect of exendin-4. (A) The canine pancreatic cells were immunohistochemically stained for insulin at 2 weeks. (B) The canine pancreatic cells of graft were stained for insulin at 8 weeks. Beta-cells percentage in the transplanted pancreatic cell graft at 2 weeks (C) and 8 weeks (D). (Original magnification ×400)
*P ≤ 0.05.
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