13 1 Vol13 No1 2009 2 Life Science Research Feb 2009 2009 PDX-1 1, 2 2*, 1 400037 2 400016 : 3, PDX-1, RT-PR PDX-1 PDX-1, PDX-1 mrn : ; PDX-1; ; :R617 : : 07-7847(2009)01-0050-05 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 400037 hina 2 Department of Hepatobiliary Surgery & Liver Transplantation First ffiliated Hospital hongqing University of Medical Science hongqing 400016 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 2009 13 1 050~054 PDX-1 2008-05-23 2008--29 04-2-121 1982- * 1963- E-mail duchengyou@126com
1 : PDX-1 51 1 11 30 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 42 30 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 -3 202 bp β-actin 80 5 -GTTTGT-3 2 14 Hank s 2 min 94 30 s 55 30 s 72 1 min % Ham s F- actin 25% Ficoll 2 ml 19 Hank s 2 ml 2 500 r / min 15 min 25% Hank s Ham s F- 15 3 % Ham s F- % 5 -TGTTGTGTGT-3 360 bp PR 94 30 72 7 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-400 2 ml DMEM/F-12 Susan onner- 11% Ficoll-400 Hank 2 ml Weir [2] 50 μl / cm 2 2 500 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 Group n Purity x ± s 8 mmol / L 28 d 494% ± 669% PR 94 3 min 95 622% ± 651% 30 s 58 30 s 73 1 min 30 735% ± 560% Notes P < 005 22 24 h 1 Ham s F- 21 3 1 DMEM / F12 48 h 494% ± 669% 622% ± 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 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-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 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 PDX-1 mrn Fig5 The expression of PDX-1 mrn after transdifferentiation 0421 1 ± 0155 4 5 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 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 < 005 750 500 3 PDX-1 250 2 000 1 000 750 500 250 0 05 1 ± 0040 4 Table 3 The comparison of surface density of PDX-1 expression after transdifferentiation in,, group Group n Surface density x ± s 0420 7 ± 0154 1 0438 3 ± 0135 8 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 0640 5 ± 0148 6 0435 4 ± 0142 2 0301 1 ± 0160 4 Notes P < 005 26 PDX-1 mrn RT-PR 5 3 0 3 β-actin PDX-1 [3] Ficoll-400 Ficoll-400 [4] K-19 PDX-1 20%~11% Ficoll 90% [5] 1 2 25% Ficoll Hank s 3 25% 11% Ficoll Hank s Ficoll 2% [6~8] 2% 0~250 bp
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