Isolation And Identification Of Cancer Stem Cells In Prostate Carcinoma Du145 Cell Line And The Investigation Of Their Biological Properties And Associated Mechanisms

Partâ… Isolation and identification of cancer stem cells in human androgen independent prostate carcinoma Du145 cell line【Objective】The aim of this study is to isolate the populations of cells with different cell surface molecules in Du145 cell line and to investigate which population of cells have the stem cell property, to identify the cancer stem cells and their markers in Du145 cell line.【Methods】To culture Du145 cells in vitro with DMEM that contain 10% FBS and to detect the expression of cell surface molecules and differentiation markers of Du145 cells with immunocytochemistry and flow Cytometric Analysis. We isolated CD117⁺ cells, CD34⁺ cells and CD44⁺ integrin. _2.1⁺CD133⁺ cells with MACS and typeâ… collagen adhesion. Clone formation assay, immunocytochemistry, flow Cytometric Analysis and CellTiter Blue proliferation assay were used to identify the cells population which had self renewal ability, unlimited proliferative potential and extensive differentiation ability.【Results】Du145 cells express CD24,CD34,CD44,CD59,CD117,CD133,CK14,CK18,Highly purified CD117⁺ cells, CD34⁺ cells,CD44⁺integrin. _2.1⁺CD133⁺ cells was harvested with MACS and typeâ… collagen adhesion. The Clone formation efficiency (CFE) of CD44⁺integrin. _2.1⁺CD133⁺ cells in Du145 cell line is higher than that of other population of cells (P＜0.05). The CFE of CD133⁺ cells was not significantly changed in the next generation. Clone generative ability of CD117⁺ cells,CD34⁺ cells is minimal and the CFE significantly decreased in next generation. The clones formed by CD44⁺integrin. _2.1⁺CD133⁺ cells are remarkably different morphologically from those formed by other populations of cells. CD44⁺integrin. _2.1⁺CD133⁺ cells present self renew and extensive differentiation potential while CD117⁺ cells,CD34⁺ cells not. Compared with integrin. _2.1^-/lowCD133^- cells and the unsorted Du145 cells, CD44⁺integrin. _2.1⁺CD133⁺ cells presented higher proliferative potential. 【Conclusion】CD44⁺integrin. _2.1⁺CD133⁺ cells presented clone generative, self renewal, extensive differentiation ability and higher proliferative potential, i. e stem cell property. CD44, integrin. ^2.1, CD133 could be the cancer stem cell markers for Du145 cells. CD117⁺ cells,CD34⁺ cells presented no stem cell property.Partâ…¡The biological properties of cancer stem cells in human androgen independent prostate carcinoma Du145 cell line and the investigation of the associated mechanism【Objective】To investigate the migration, invasion and tumorigenic abilities of CD44⁺integrin. _2.1⁺CD133⁺,·_2.1^-CD133^- and the unsorted Du145 cells and to study the mechanisms that involved in maintenance of the stemness of the cancer stem cells.【methods】To investigate the in vitro migration and invasion abilities of CD44⁺ integrin. _2.1⁺CD133⁺ and the unsorted Du145 cells by TransweU chamber chemotaxis and chemoinvasion assay and to detect the in vivo tumorigenic abilities of CD44⁺ integrin. _2.1⁺CD133⁺, integrin. _2.1^-CD133^- and the unsorted Du145 cells with NOD/SCID mice xenograft Model. We also studied the expression level of several associated genes in integrin. _{2. 1}⁺CD133⁺ cells and the unsorted Du145 cells.【Results】Compared with the unsorted Du145 cells, the migration and invasion abilities of CD44⁺integrin. _2.1⁺CD133⁺ cells were promoted(P＜0.05). The results of xenograft Model indicated that a small number of grafted CD44⁺ integrin. _2.1⁺CD^- 133⁺ cells can form a new tumor. It is difficult for integrin. _{2. 1}^-CD133^- and the unsorted Du145 cells to form a new tumor even though more cells were grafted(P＜0.05). The expression of bcl-2, bax, fas, c-myc, survivin and. -catenin was examined by Real-Time PCR, with Bcl-2 null in both CD44⁺integrin. _2.1⁺CD133⁺ cells and the unsorted Du145 cells. The relative expression levels of c-myc and. -catenin in CD44⁺integrin. _2.1⁺CD133⁺ cells was up regulated by 202.6% (180%～220%) and 309.8% (270%～350%), respectively. Bax was down regulated by 63.6% (58%～68%). Fas and survivin expression showed no significant difference in both cell populations.【Conclusion】Prostatic cancer stem cells in Du145 cells presented promoted migration, invasion and tumorigenic abilities. Bax, c-myc and. -catenin may play a role in maintenance of their stermness. Partâ…¢gene expression profiling in cancer stem cells and the total population of cells in Du145 cell line with microarray【Objective】To screen for genes related to prostate cancer stem cells in Du145 cell line by comparing the differences in the expression profile between prostate cancer stem cells and the unsorted Du145 cells.【methods】CD44⁺integrin. _2.1⁺CD133⁺ cells and the unsorted Du145 cells were harvested with MACS and typeâ… collagen adhesion. The mRNA expression level of CD44⁺integrin. _2.1⁺CD133⁺ cells and the unsorted Du145 cells was detected with microarry that was provided by Affymetrix. The genes that were studied in the experiment included 47, 000 genes and mutants. The genes with different expression level subjected to hierarchical cluster analysis with the microsoft GCOS1.2 according to their functions. The accuracy of the results of microarray was judged by the coincidence of the results and that of Real Time PCR.【Results】231 genes with a different expression level were screened out. Compared with the Du145 cells, the expression level of 138 genes was elevated and that of 93 genes was down regulated in CD44⁺integrin. _2.1⁺CD133⁺ cells. The genes included Apoptosis or tumor associated genes, binding, Cell cycle, metabolism, enzyme regulator activity, signal transducer activity and transcription regulator activity associated genes. We compared the results of Real Time PCR and microarry. The results of microarry and Real Time PCR are nearly coincident.【Conclusion】Microarray could screen for genes related to prostate cancer stem cells in Du145 cell line effectively and accurately. Cancer stem cells may maintain their distinct biological properties through multiple channels, regulation of multiple genes or nets of genes.