PDX-1. The Expression of PDX-1 mrna in Different Pancreatic Islets Purity that Contain Stem Cells Before and After Transdifferentiated Culture

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1 13 1 Vol13 No Life Science Research Feb PDX-1 1, 2 2*, : 3, PDX-1, RT-PR PDX-1 PDX-1, PDX-1 mrn : ; PDX-1; ; :R617 : : (2009) The Expression of PDX-1 mrn in Different Pancreatic Islets Purity that ontain Stem ells efore and fter Transdifferentiated ulture WNG Wei-wei 1 WNG Ji-ming 2 DU heng-you 2* 1 Department of Hepatobiliary Surgery Xinqiao Hospital of Third Military Medical University hongqing hina 2 Department of Hepatobiliary Surgery & Liver Transplantation First ffiliated Hospital hongqing University of Medical Science hongqing hina bstract Three concentrations of islets in rats were purified by three different methods Then the pancreatic stem cells were purified and transdifferentiated The pancreatic stem cells were identifed with rabits to rats antibody PDX-1 The expression of PDX-1 in pancreatic stem cells pre and post transdifferentiation were tested by immunocytochemistry and RT-PR The results showed that after transdifferentiation the expression of protein PDX-1 in cell nuclear and PDX-1 mrn were increased This indicated that the daughter cells of the pancretic stem cells after transdifferentiation still maintain the character of stem cells Key words donor islets PDX-1 rat stem cells Life Science Research ~054 PDX * duchengyou@126com

2 1 : PDX SD 180~200 g 36 h V Ham s F- 37 Sigma DTZ Ham s F- 5% O 2 95% O 2 14 d 2 d Trizol DMEM / F12 15 d Gibco Matrigel 70% D S 5 5 ROHE KGF PEpro tech PDX-1 16 Upstate SP PDX-1 RT_PR PDX-1 17 PDX-1 β-actin PDX-1 SP 12 D Olympus PS SIGM PR Thermo Hybaid 5 13 Hesseu 18 PDX-1 mrn [1] SD Ham s F- 14 d PS 2 4 ml 1 ml Trizol min 99 5 min 5 5 min 50 ml Falcon cdn PDX-1 5 -GGGGTG 37 25~30 min T-3 5 -GTTGTGTT 4 Hank s GTT bp β-actin GTTTGT Hank s 2 min s s 72 1 min % Ham s F- actin 25% Ficoll 2 ml 19 Hank s 2 ml r / min 15 min 25% Hank s Ham s F % Ham s F- % 5 -TGTTGTGTGT bp PR min Ham s F- μg/l KGF ITS Hank s 5 mg/l 5 mg/l 5 mg / L % Ham s F- 2 g / L S mmol / L 25% Ficoll ml DMEM/F-12 Susan onner- 11% Ficoll-400 Hank 2 ml Weir [2] 50 μl / cm r / min 15 min 1 Matrigel 11%~25% O 2 95% O 2 37 Hank s % 2 d 28 d

52 2009 1 PDX-1 1 PDX-1 17 111 PDX-1 mrn KGF S ITS DMEM/F12 72 7 min 112 ± x ± s 3 Ham s F- SPSS130 2 1 Table 1 The comparison of islets purity gained by different purification methods

± 651% 735% ± 560% P < 005 1 1 () DMEM / F12 ( ) (40 ); () Ham s F- (40 ); () Fig1 The morphology of pancreatic stem cells pre and post transdifferentiation The adherent cells in group

serumfree DMEM/F12 medium 23 PDX-1 3 3 2 3 PDX-1 2 (40 ) () PDX-1 (40 ); () Fig2 ells positive for dithzthone staining(40 ) PDX-1 (40 ) Fig3 The results of PDX-1 immunohistochemistry

3 PDX-1 1 PDX PDX-1 mrn KGF S ITS DMEM/F min 112 ± x ± s 3 Ham s F- SPSS Table 1 The comparison of islets purity gained by different purification methods Group n Purity x ± s 8 mmol / L 28 d 494% ± 669% PR 94 3 min % ± 651% 30 s s 73 1 min % ± 560% Notes P < h 1 Ham s F DMEM / F12 48 h 494% ± 669% 622% ± 651% 735% ± 560% P < () DMEM / F12 ( ) (40 ); () Ham s F- (40 ); () Fig1 The morphology of pancreatic stem cells pre and post transdifferentiation The adherent cells in group after cultured with serum-containing medium 40 The morphology of cells in group after cultured with serum-free Ham s F- medium 40 The morphology of cells in group after cultured with serumfree DMEM/F12 medium 23 PDX PDX-1 2 (40 ) () PDX-1 (40 ); () Fig2 ells positive for dithzthone staining(40 ) PDX-1 (40 ) Fig3 The results of PDX-1 immunohistochemistry stain 24 before and after transdifferentiation PDX-1 The PDX-1 were expressed in cytoplasm before transdifferentiation 40 The PDX-1 were expressed 3 2 in cyto nuclear after transdifferentiation 40

1 : PDX-1 53 2 PDX-1 mrn Table 2 The comparison of surface density of PDX-1 expression before transdifferentiation in,, group 5 M Group n Surface density x ± s bp 0233 1 ± 00157 0114 3 ± 0021 5 5

transdifferentiation in, PDX-1 mrn, group Group RT-PR 2% n Optical density x ± s 0~250 bp 4 0782 8 ± 0134 1 0758 3 ± 0147 7 PDX-1 mrn 0745 8 ± 0150 9 4 Notes P > 005 M bp 2 000 1 000 Notes P < 005

4 1 : PDX PDX-1 mrn Table 2 The comparison of surface density of PDX-1 expression before transdifferentiation in,, group 5 M Group n Surface density x ± s bp ± ± PDX-1 mrn Fig5 The expression of PDX-1 mrn after transdifferentiation ± PDX-1 mrn Notes P > 005 Table 5 The comparison of integral optical density of 25 PDX-1 mrn expression after transdifferentiation in, PDX-1 mrn, group Group RT-PR 2% n Optical density x ± s 0~250 bp ± ± PDX-1 mrn ± Notes P > 005 M bp Notes P < PDX ± Table 3 The comparison of surface density of PDX-1 expression after transdifferentiation in,, group Group n Surface density x ± s ± ± β-actin PDX-1 4 PDX-1 mrn Fig4 The expression of PDX-1 mrn before transdifferentiation 4 PDX-1 mrn Table 4 The comparison of integral optical density of PDX-1 mrn expression before transdifferentiation in,, group Group n Optical density x ± s ± ± ± Notes P < PDX-1 mrn RT-PR β-actin PDX-1 [3] Ficoll-400 Ficoll-400 [4] K-19 PDX-1 20%~11% Ficoll 90% [5] % Ficoll Hank s 3 25% 11% Ficoll Hank s Ficoll 2% [6~8] 2% 0~250 bp

5 (References): PDX-1 Lu J [6] in the pig[j] Transplant Proc PDX-1 PDX-1 Proc Natl cad Sci US Ham sf- 14 d humans years of experience[j] PDX-1 [4] DMEM / F12 PDX-1 K-19 PDX-1 mrn Todorov I 4~7 d K-19 PDX-1 Organ Transplantation [9] 90% Ficollgradients[J] Transplant Proc ~7 d PDX-1 terol KGF PDX-1 Sharma [] PDX-1 RT-PR 3 PDX-1 mrn 3 PDX- 1mRN natomy KGF KGF PDX-1 [11,12] [1] HESSEU J MEYER G P WEYER J et al In situ intraductal collagenase injection for preparation of langerhans [2] ONNER-WEIR S TNEJ M GORDON et al In vitro cultivation of human islets from expanded ductal tissue [J] [3] RNDHORST D RNDHOST H HERING J et al Islet isolation from the pancreas of large mammals and Exp lin Endocrinal [J] YNG huang WNG Ji-ming DU heng-you et al mrn expression of stem cell markers K-19 and PDX-1 in the different purity of islets in rats[j] hinese Journal of [5] OLK SWNSON Islet purification using Euro [6] LU J GU Y P XU X et al dult islets cultred in collagen gel transdifferentiate into duct-like cells[j] World J Gastrolen- [7] TODOROV I OMORI K PSUL M et al Generation of human islets through expansion and differentiation of non-islet pancreatic cells discard pancreatin discard after islet isolation[j] Pancreas [8] [J] SHI Kai XU Li DENG Yi-hao et al Study on culture method of pancreatic ductal epithelial cells from kunming mice[j] Sichuan Journal of [9] TODOROV I OMORI K PSUL M et al Generation of human islets through expansion and differentiation of non-islet pancreatic cells discard pancreatin discard after islet isolation[j] Pancreas [] SHRM ZNGEN D H REITZ P et al The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration[j] Diabetes [11] GEMYR V KERR-ONTE J ELIH S et al duclt human cytokeration19-postive cells reexpress insulin promoter factor in virtro futher evidence for pluripotent pancreatic stem cells in human[j] Diabetes [12] HOSOTNI R ID J KOGIRE M et al Expression of pancreatic duodenal hoemobox-1 in pancreatic islet neogenesis after surgical wrapping in rats[j] Surgery

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