Study Of Biological Characteristic And Regulation Of Differentiation And Proliferation Of Tumor Stem Cells In Pancreatic Adenocarcinoma

Part I Isolation,Culture and identification of tumor stem cells in Pancreatic AdenocarcinomaObjective The purposes of this research were to explore the method of Isolation,Culture and identification of tumor stem cells in Pancreatic Adenocarcinoma.Methods The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 (1:1 volume) (DMEM-F12) supplemented with 20% fetal bovine serum(FBS). Subpopulation cells which have properties of tumor stem cells were isolated according to the cell surface markers CD44 and CD24 by flow cytometry from pancreatic adenocarcinoma cell line PANC-1. The tumors growths of different subpopulation cells which injected into the hypodermic of right and left armpit of nude mice were studied, and expression of CD44 and CD24 of the CD44+ CD24+ cells -formed nodules and PANC-1 cells were detected by streptavidin-perosidase immunohistochemical staining. And CD44 and CD24mRNA were detected by RT-PCR.Results In DMEM/F12 supplemented with 20% FBS, 5.1% to 17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 21.8% to 70.1% expressed CD24, only 0.9% to 3.5% of cells were CD44+CD24+;but in serum-free medium ,they were 5.9% to 16.8%,19.5% to 74.6% and 0.8% to 4.1% .The sorted cell population with the highest tumorigenic potential were those cells expressing CD44 and CD24. Implantation of 1×105 CD44-CD24-cells in nude mice, no tumor growth was evident at 12 weeks. In contrast, nude mice implanted with 5×103 CD44+ CD24+ cells had large tumors evident at 4 weeks(2/8), at least a 20～50-fold increased in tumorigenic potential(P<0.05 or P <0.01). There was no obvious histological difference between the cells of the CD44+ CD24+ cells -formed nodules and PANC-1 cells.Conclusion CD44 and CD24 may be used as the cell surface markers for isolation pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44+CD24+ have properties of tumor stem cells.Part II Biological Analysis of Tumor Stem Cell of Pancreatic AdenocarcinomaObjective To study biological characteristic of tumor stem cell of pancreatic adenocarcinoma.Methods The proliferative capability of tumor stem cell in vitro were estimated by MTT method. The cell cycles and DNA content of these cells were investigated using flow cytometry, and the telomerase activity was detected by telomeric repeat amplification protocol(TRAP).Results Compared with CD44-CD24-cells, CD44+CD24+ cells had a lower growth rate in vitro. For the former, index growth trend appeared at 5th day, while the latter appeared at 7th day. The telomerase activity of CD44+CD24+ cells was higher, compared with CD44-CD24-cells. The percentage S/G2/M cells of CD44+,CD24+ and CD44+CD24+ was lower , compared with CD44-,CD24- and CD44-CD24- cells(P<0.05 or P<0.01).Conclusion Tumor stem cells of pancreatic adenocarcinoma have properties of a lower growth rate in vitro, higher telomerase activity and relative stationary. Part III Regulation the differentiation and proliferation of tumor stem cell of pancreatic adenocarcinoma by Notch1Objective To study regulation the differentiation and proliferation of tumor stem cells of pancreatic adenocarcinoma by activated Notch1.Methods The rhNF-κB, an activator of Notch,and theγ-secretase inhibitor II(MW167),were added into the mediums of tumor stem cells of pancreatic adenocarcinoma respectively,and the control was added with PBS buffer.Then the detection were carried out by RT-PCR,immunohistochemistry, Western Blot and flow cytometry.Results Notch1 and JAG1 were expressed in tumor stem cell of pancreatic adenocarcinoma cells.The expressions of CD44 and CD24 of rhNF-κB group were more than those of control,and the effect of promoting proliferation was obvious P<0.05或P<0.01),more cells seayed at S and G1 phase. But the expressions of CD44 and CD24 ofγ-secretase inhibitor II group were decreased apparently.Conclusion When Notch signaling was activated, the tumor stem cells of pancreatic adenocarcinoma were going on proliferating.On the contrary, the cells were going on differentiating when Notch signaling was suppressed.