Preliminary Study Of Isolation And Identification Of Murine Cancer Stem Cell From Murine Melanoma

Objective: Recent studies suggest that cancer can arise from a cancer stem cell (CSC), a tumor-initiating cell that has properties similar to those of stem cells. CSC has been identified in several human malignancies, however, it is not reported yet that there are CSC isolated and identified from animal cancer cell. According to the many similar biological features between the cancer cell of human origin and the cancer cell of animal origin, we try our best to isolate and identify murine CSC from the murine melanoma B16F10 cells, and to lay a foundation for research of resistance to chemotherapeutic agents in the CSC, target therapy for CSC, and signaling pathway in carcinogenesis etc in future.Methods: Isolating B16F10 cells from the murine melanomas model and selecting the specific marked B16F10 cell from the cells binding different monoantibody labeled with immune magnetic beads by magnetic activated cell sorting system (MACS), the purity of isolated specific marked B16F10 cells was valuated by flow cytometry (FCM) further, and the proliferation activites of different CD phenotypes B16F10 cells were observed in the cultured B16F10 cells by keeping count of cells in vitro. After the capability of clonegenesis were selected in soft agar culturing or common culture, the potential clonegenesis B16F10 cells with the specific CD phenotypes were inoculated s.c into C57BL/6 mice and the tumorigenic process were compared with the different CD phenotypes B16F10 cells in C57BL/6 mice. The compostive analysis and identification CSC was finally made from a series of experiments results.Results: 1.CD133~+, CD133~―, CD44~+, CD44~―, CD44~+CD133~+,CD44~+CD133~―B16F10 cells were successfully isolated from the murine melanomas cells binding different monoantibody labeled with immune magnetic beads by MACS and these specific marked B16F10 cells were basically consistent with the identified B16F10 cells by FCM. The FCM analysis results suggest that the specific CD phenotypes B16F10 cells are a high purity also. 2. The proliferation activity of CD133~+ phenotype B16F10 cells is stronger than those of CD133~―phenotype B16F10 cells and wild B16F10 cells in vitro. 3. The results of soft agar culture or common culture show that the rate of clonegenesis of CD133~+ phenotype B16F10 cells are prior to CD133~― phenotype B16F10 cells and the rates of clonegenesis of CD44~+, CD44~+CD133~+ phenotypes B16F10 cells are prior to CD44~―, CD44~+CD133~―phenotypes B16F10 cells respectively. 4. The tumorigenesis of CD133~+ phenotype B16F10 cells in...