Objective To investigate the effects on self-renewal of pancreatic cancer stem cells by inhibiting hedgehog signaling pathway through cyclopamine. Methods PANC1 stem cells, PANC1 adherent cells and immortalized pancreatic ductal epithelial H6C7 cells were treated with 0.5, 1, 2, 5, 10 mol/L of cyclopamine for 24, 48, 72 h. The expression of Smo mRNA and Gli1 mRNA were detected by real-time PCR.Cell growth viability was measured by CCK 8. Cell cycle and apoptosis were determined by flow cytometry.Results Seventy-two hours after cyclopamine treatment, the Smo mRNA expressions of PANC1 stem cells,PANC1 adherent cells and H6C7 cells were 1,0.83 and 2.61; the expressions of Gli mRNA were 57.27,26.35,1; the inhibitory rates were ( 37.85 ± 13.69 ) %, ( 8.53 ± 4.43 ) %, (43.55 ± 28.98 ) %. Compared with PANC1, the expressions of Smo mRNA, Gli1 mRNA and the inhibitory rate of PANC1 stem cells significantly increased ( P ＜ 0.05 ). The proportion of G1 stage of PANC1 stem cells significantly decreased from (67.41 ±6.35)% to (36.53 ±6.03)% (P ＜0.05), and the apoptosis decreased from (10.95 ±5.68) % to ( 5.73 ± 1.42 ) % ( P ＞ 0.05 ). The proportion of G1 stage of PANC1 cells significantly decreased from ( 67.64 ± 6.88 ) % to ( 53.13 ± 1.10 ) % ( P ＜ 0.05 ); the apoptosis decreased from ( 12.08 ±4.12)% to (5.66 ± 1.33)% (P ＞0.05). While both the proportion of G1 stage and apoptosis of H6C7 cells was not significantly different. Conclusions Cyclopamine can inhibit the proliferation of PANC1 stem cells via blocking hedgehog signal pathway, and the mechanism may not be associated with cell apoptosis.

Objective To establish the method of suspension culture for stem cells from human pancreatic cancer cell line PANC1.Methods PANC1 cells were cultured in serum-free medium under floating-culture system.Tumor cell spheres were observed by optical microscope.Expression of CD133 and cell cycle were detected by flow cytometry.Cancer stem cells were induced to differentiate with 10%FBS,and expression of CK18,was evaluated with immunofluorescence microscope.Spheres cells were injected into the subcutaneous space of NOD/SCID rat and tumor formation was monitored weekly.Results PANC1 cells could form the stem cells spheres,and the rate of sphere formation was stable between 4%0 and 5%0 after 20 passages in vitro.The expression of CD133(5.91±0.7%)and proportion of G0/G1 phase cell(80.99±2.60%)was significantly increased in spheres cells compared with parental PANC1 cells(1.44±0.52%and 69.01±5.03%),and the difference was statistically significant(P＜0.05).When these spheres cells were cultured in media with serum,these cells gradually returned to the status of parental cells and expressed CK18,2×103 sphere cells injection could initiate tumor fornmtion in NOD/SCID rat.Conclusions Tumor spheres stem cellscould be generated under serum-free floating-culture system.The sphere cells possessed the capacities of self renew,difierentiation,and tumorigenic potential.

In the current work, the fusion gene including somatostatin (SS) and the hepatitis B surface antigen gene was cloned into a balanced lethal system plasmid (pYA3493), and then transformed into asd- attenuated Salmonella choleraesuis C500 strain, the positive transformant without antibiotic resistance gene was confirmed by restriction analysis and DNA sequencing, designated as pYA-SS. The expression and immunogenicity of fusion protein were detected by SDS-PAGE and Western blot analysis. These results show that the recombinant prokaryotic expression plasmid pYA-SS could express the SS fusion protein with good immunogenicity in C500 strain. In above all, this study could provide reliable materials to develop novel, good and safe vaccine in enhancing the growth of animals.
Animals ; Artificial Gene Fusion ; Cloning, Molecular ; Hepatitis B Surface Antigens ; genetics ; Humans ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Salmonella arizonae ; genetics ; metabolism ; Somatostatin ; biosynthesis ; genetics ; immunology