| In our nation, the genetic resources of domesticated animals are abundant, while theirconservation is far from optimism. Stem cell possesses the renewability and multipotency andmeets the requirements of storing, which make it a seeding cell to preserve the genetic resourcesfrom livestock and poultry with a great significance. In this work, we established a chickenpancreatic stem cell line, then induced them differentiated into beta cells which could produceinsulin, analyzed the difference of expression profile between the natural differentiated beta cellsand induced ones via gene chip technology and explored the differentiation mechanism. Results:1. The method of enzymic digestion and differential attachment was suitable to derivedpancreatic stem cells in vitro and cell were cultured for3passages, the Nestin+cells sorted byflow cytometry were generated to passage23. The majority of cells presented clone-like growth,the nuclei were round or in kidney-like shape, no significant difference of cell viability before andafter cryopreservation（p>0.05）and the proliferation ability was strong, the pancreatic stem cellspecific surface makers Pdx-1and Nestin were detected by immunofluorescence,flow cytometryand RT-PCR, resulted positively, which suggested the optimized culture system was appropriatefor the cell line, the biological characterization was unique to pancreatic stem cell.2. The induced beta cells displayed typical vesicles and stain positively by dithizone, andthey were proved the ability of insulin secretion by glucose stimulation experiment, the analysis ofcalcium ion concentration by maker Fluo-4/AM also demonstrated the secretion activities, thesesuggested a success induction.3. The gene chip showed there are20signal paths concentrating distributed and10areclosely related to the development of beta cells, multiple transcript factors at different paths couldregulated in some sense,46differential genes were discovered among Wnt, Notch, TGFβ andPDGF signal paths including24up-regulated genes and22down-regulated genes, Real time PCRresults showed that only gene DLC1, FZD3and SFRP2was not accordance with gene chipanalysis, the coincide rate was95.6%, besides, JUN, VAV2, MAPK13, BMP6and WNT5A werehighly significant down regulated, gene DLL1highly significant up-regulated. These genes mayplay a key role or significantly regulated by other genes in the differentiation process from PSCsinto pancreatic β cells.In this study, we obtained a stable chicken pancreatic stem cell line and accomplished thecharacterization, we gained the expression profile of β cells via gene chip and found46relativegenes, the dynamic expression of those genes was accessed by Real time PCR and the expressionpattern was expounded. |
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