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Destination rat mesenchymal stem cells in vivo tumorigenicity model biological characteristics and tumorigenic a monoclonal cancer cell lines (K3); establish the K3 tumor stem cells (K3-SP) separation, identification methods; preliminary Cell Biology molecular biology techniques to explore the K3 cancer stem cells and non-cancer stem cell biological characteristics and gene expression; enrichment of the K3 tumor stem cells of serum-free culture. Growth curve, flow-surface markers, PCR, immunohistochemistry and other techniques to study the biological characteristics of K3 cells. Preparation of K3 cell suspension after Hoechst 33342 fluorescent staining, flow cytometry analysis and elect SP subsets. Using five kinds of training methods to train K3: 5? S 10? S, serum-free, serum-free plus three K3 and K3 cells in BALB / c nude mice tumorigenic growth factor culture. To obtain a sufficient amount of K3 cells, tumor tissues than 400 mesh copper mesh to obtain a single cell suspension. The single cell suspension after Hoechst33342 fluorescent dye selected K3-SP cells and K3-NSP-cells using flow cytometry. BALB / c nude mice tumorigenicity experiment and observation comparing tumor volume size sorting get K3-SP cells and K3-NSP cells. Use Crystal Core ? rat genome oligonucleotide microarray Compare K3-SP/K3-NSP cell gene expression differences using fluorescence real-time quantitative RT-PCR validation of differentially expressed genes. Compare K3-SP induced tumor tissue and K3-SP tumorigenic organization of ABCG2, of OCT4, PCNA, P53 immune staining results. The serum-free culture enrichment K3-SP cells. Result of K3 of tumor cells expressing rat mesenchymal stem cell surface antigens such as CD29, CD44, CD90, weak expression of CD45; K3 tumor cells with normal tumor cells expressed genes and growth characteristics, tumorigenicity in BALB / c nude; K3 tumor cell heterogeneity, while containing a small amount of cancer stem cells. Different fetal bovine serum concentration culture K3 cells K3-SP cells content differences, 5? S culture and 10? S culture analysis K3-SP content of 1.01% and 0.73%, respectively. K3-SP represents K3-NSP Blab / c nude mice, tumor formation rate was significantly enhanced, 5 × 10 to 3 K3-SP cells can nude tumorigenicity, and its tissue types fibrosarcoma. Microarray analysis, K3-SP and K3-NSP 56 differentially expressed genes, which is up 40, down 16, Real-timePCR verify T-Bx3, Mafb, P1k2 downregulated genes up-regulated genes and Vegf a. Immunohistochemistry showed that K3-SP the tumorigenic organization for P53 Pcna Abcg2 Oct4-positive, while negative the K3-NSP tumorigenic tissue. K3 showed the growth of suspended spherical cells in serum-free medium containing bFGF, EGF, LIF three factors without the medium of the three factors, Real-time PCR showed Oct4 expression were two serum-free medium differences. Serum-free medium plus three factors after 15 days of culture, the the SP content reached 7.73%, having a significant enrichment K3-SP role. Mutated tumorigenic conclusion K3 tumor cells from the bone marrow MSC, which contains K3-SP cells K3-SP cells K3-NSP cells there are obvious differences in the tumorigenic gene expression, K3-SP cells with strong tumorigenic ability. Serum-free the plus factor cultivate significant enrichment K3-SP, these studies find K3-SP cell-specific molecular markers and the in-depth study of the K3-SP to provide experimental evidence.
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