| Background and Objective:Pancreatic adenocarcinoma is a highly lethal disease,which is usually diagnosed in an advanced state for which there are little or on effective therapies.It has the worst prognosis of any major malignancy(3%5-year survival).Along with deep research on the stem cells and the tumor origin,people realized that stem cell's dysdifferentiation and the multiplication can cause the occurrence of the tumor.There are very complicated relations between the tumor origin,malignancy,drug resistance,metastasis,reccurence and the unusual expression of stem cell genes.The transcription factors Oct4,nanog combined with Sox2 play an important role in embryo development and embryonic stem cell differentiation,and have been recognized as fundamental in the maintenance of pluripotency in embryonic stem cells and in primordial germ cells.Manipulation of the expression of Oct4 and Nanog can initiate the transition among these stem cells.Recent research showed that the adult stem cell expressed Oct4 might be the origin of malignant tumors.Several studies have demonstrated that Oct4 was expressed by some solid cancer cell lines,like MCF-7,Hela. However,the expression of Oct4 on pancreatic cancer cell lines has not been studied systematically.The mechanisms and the effect of Nanog on solid cancer cells has not been declared yet.In the present study,we searched the exression of Oct4 and Nanog in both mRNA and protein levels on three pancreatic cancer cell lines with different differential degrees (Bxpc-3,Pane-1 and Mia PaCa-2).Single-cell clonal culture was used to determine the percentage of cells with self-renewal capacity.And the expression of Oct4 and Nanog on normal cultured,single-cell cloned and subcloned Panc-1 were compared by real-time PCR.Materials and Methods:1,A rapid and reliable real-time quantitative reverse transcription polymerase chain reaction(real time RT-PCR) approach using LightCycler Technology was used to detect the expression of Oct4 and Nanog on three pancreatic cancer cell lines with different differential degrees(Bxpc-3,Panc-1 and Mia PaCa-2) in mRNA levels.2,Western blot was used to detect the expression of Oct4 and Nanog on three pancreatic cancer cell lines with different differential degrees(Bxpc-3,Panc-1 and Mia PaCa-2) in protein levels.And the in situ expression of Oct4 and Nanog was examined by immunofluorescence.3.Single-cell clonal culture was used to determine the percentage of cells with self-renewal capacity.And the expression level of Oct4 and Nanog on normal cultured, single-cell cloned and subcloned Panc-1 were compared by real-time PCR.Results:1,In real time RT-PCR examination,all the three pancreatic cancer cell lines with different differential degrees(Bxpc-3,Panc-1 and Mia PaCa-2) express the mRNA of Oct4 and Nanog.Compared to GAPDH,theΔCT of Oct4 and Nanog in Bxpc-3 were 10.64±1.23 and 16.44±1.01 respectively;theΔCT of Oct4 and Nanog in Panc-1 were 10.64±1.23 and 16.44±1.01 respectively;theΔCT of Oct4 and Nanog in Bxpc-3 were 10.74±1.88 and 15.34±2.43;theΔCT of Oct4 and Nanog in Mia PaCa were 8.98±0.73 and 14.44±2.17 respectively.2,The expression of Oct4 and Nanog in protein was confirmed by western blot.And the immunofluorescence confirmed the location of Oct4 and nanog just like other researches before.3,About 22.7%(5/22) Panc-1 cells were capable of forming clonal cell spheres,and 26.7%(4/15) can form subclonal cell spheres.In real time RT-PCR examination,the expression of Oct4 and Nanog among normal cultured,single-cell cloned and subcloned Panc-1 had no statistically difference.Compared to GAPDH,theΔCT of Oct4 in single-cell cloned and subcloned Panc-1 were 10.51±0.58 and 9.91±0.98,while theΔCT of nanog were 14.61±2.92 and 14.2±1.04 respectively.Conclusion:1,All three pancreatic cancer cell lines with different differential degrees(Bxpc-3, Panc-1 and Mia PaCa-2) express the mRNA and protein of Oct4 and Nanog.2,In all the three pancreatic cancer cell lines with different differential degrees(Bxpc-3, Panc-1 and Mia PaCa-2),Mia PaCa-2 expressed higher level of Oct4 than Panc-1 and Bxpc-3,and higher level of nanog than Bxpc-3.Since Mia PaCa-2 is much more malignant than Bxpc-3,it indicates that the expression level of Oct4 and Nanog might connect with the differential degree and malignancy of pancreatic cancer.3,There was no statistically difference on the expression of Oct4 and Nanog among normal cultured,single-cell cloned and subcloned Panc-1 cells in real time RT-PCR examination.
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