The Selection And Functional Verification Of Mice Pancreatic Cancer Stem Cells From The MPC-83 Cell Line

Objective:To select the mice cancer stem cells from MPC-83 Cell Lines and identify the biological characteristics. To study the drug-resistance ability of the mice cancer stem cells. This experiment provides the basis of the further research on immunosuppressive function in mice.Methods:1.Flow cytometry assay was applied to select the CD24+CD44+ pancreatic cancer stem cells from the MPC-83 cell line.2.The sorted cells were cultured in the serum-free medium to observe the ability of self-renewal.3.The ability of migration and invasion was detected by wound healing assay, transwell migration and invasion assay.4.The oncogenicity of cancer stem cells were performed by the mouse transplantation tumor erperiment.5.Flow cytometry assay was used to detect the cell cycle of the cancer stem cells.6.CD24+CD44+MPC-83 cancer stem cells and normal MPC-83 were treated with gemcitabine, and the viability and sensitivity was performed by the MTT assay.Results:1. The CD24+CD44+ MPC-83 CSCs which were selected through the flow cytometry assay made up 5% of the total number of cells.2.The CD24+CD44+ MPC-83 CSCs survived, passaged and formed spheres in the serum free medium. The separated cells were successfully cultured and passaged in vitro, and were of self-renewing ability.3.The mean number of migrated cells of CD24+CD44+MPC-83 CSCs and MPC-83 was (145.2 ±15.4) and (112.3±11.5). The mean number of invased cells of CD24+CD44+MPC-83 CSCs and MPC-83 was (62.8±12.7) and (48.7±21.5). The data stated that the CSCs have higher migratory and invasive ability in contrast with those parental cells.4.As few as 1 × 103 cells in could initiate subcutaneously transplanted tumor in mice, which demonstrated the tumorigenicity of CD24+CD44+ MPC-83 CSCs was 100 times that of the parental cell lines.5. The cell cycle results revealed that the proportion of G0/G1 was (76.8±2.57)% in CD24+CD44+ MPC-83 CSCs,which was significant different from the proportion of (47.28±1.31)% in MPC-83 cells.6. The survival rate of normal MPC-83 cells treated with GEM decreased more sharply than that of CD24+CD44+ MPC-83 CSCs treated with GEM in the same concentration and same time.Conclusion:The selected CD24+CD44+ MPC-83 CSCs have stem cell properties and drug resistance. The research could provide the basis of the further research on individual therapy and immunosuppressive function.