The Expression Of PDX-1 In Different Pancreatic Islets Purity That Contain Stem Cells Before And After Transdifferentiated Culture

Objectives: To obtain the stem cells in different pancreatic islets purity utilizing free-serum culture medium and investigate the PDX-1 mRNA expression distinction between before and after transdifferentiate culture.Methods: 30 male SD rats (islet donors) were randomly divided into 3 groups. Pancreatic islets were isolated by injection of collagenase into pancreatic ducts, and a discontinuous Ficoll solution was applied to the purification of the islets. Three groups of different pancreatic islets number were obtatined by the following: Group A(N=10):Dissociated cell preparations were not purified;Group B(N=10): Islets sediments were purified with Ficoll (25%) gradient density;Group C(N=10): Islets sediments were purified with Ficoll (25%,11%) gradient density. maintaining the different pancreatic islets purity in serum Ham's F-10 for 36h. Then keeping the cells in free-serum Ham's F-10 for 14 days.The last stage is shift culture media from free-serum Ham's F-10 to free-serum DMEM/F12 that contains KGF,BSA,glucose and ITS. The mRNA of PDX-1 were amplified by RT-PCR, and the PDX-1 positive cells was investigated by immunocytochemistry.Results: (1) Three different islet yields were produced by continuous digestion and isolation methods, group A: 757.4±63.60;group B: 697.6±51.30; group C: 663.4±43.98. Group B vs group C, no differences were observed(P﹥0.05), but group A vs group B or group C, there were obvious statistically signification (P﹤0.05). (2) Three different islets purity were also produced by distinct purification methods. group A: 49.4±6.69%;group B:62.2±6.51%;group C:73.5±5.60%。It was obvious statistically signification in each group(P﹤0.05). (3) Expression of PDX-1 was investigated by immunocytochemistry, before transdifferentiation group A: 0.2331±0.0157, group B: 0.1143±0.0215, group C: 0.1051±0.0404. There is obvious statistically signification in each group(P>0.05);After transdifferentiation, group A: 0.4207±0.1541, group B: 0.4383±0.1358, group C: 0.4101±0.1554. There is no obvious statistically signification in each group(P>0.05) (4) The mRNA of PDX-1 expression before transdifferentiation were investigated by RT-PCR,group A: 0.6405±0.1486;group B:0.4354±0.1422;group C:0.3011±0.1604。There is statistically signification in each group(P﹤0.05). (5) The mRNA of PDX-1 expression after transdifferentiation were investigated by RT-PCR, group A: 0.7828±0.1341 ; group B :0.7483±0.1477 ; group C : 0.7358±0.1509.There is no statistically signification in each group(P>0.05)Conclusions: (1) Three methods of distinct purification have been taken to obtain different pancreatic islets purity, and each group has pancreatic stem cells. (2)The pancreatic stem cells could survive in free-serum media. (3) The pancreatic stem cells have proliferating property in non-KGF culture fluid ,but could not form new islets; Otherwise, there is new islets. (4) Before transdifferentiation, the lower pancreatic islets purity, the expression PDX-1 mRNA is more obvious; But there is no difference in each group after transdifferentiation. And after transdifferentiation ,the PDX-1 mRNA is more obvious than before.(5)The daughter cells of pancreatic stem cells in different islets purity have character about stem cells after transdifferentiation;KGF may have effect on promoting the expression of PDX-1.