| Objective To isolate the cancer stem cells(CSCs) in F6 cell line transformed from fetal bone marrow mesenchymal stem cells(FMSC), establish a method of optimizing the long time culture conditions of F6 CSCs,and identify their biological characteristics.To search for the different expression genes between F6-CD133+ cells and F6-CD133-cells,F6 and FMSC,select the new target for therapy.To investigate the methylation status of death-associated protein kinase(DAPK),fragile histidine triad(FHIT) gene promoter in FMSC and F6 cells,and explore the effect of 5-Aza-CdR on the growth of FMSC and F6 cells.Methods F6 cells were injected into BALB/c nude mice.After forming tumor,the tumor tissues were collected and dissociated to get the single cells.F6-CD133+and F6-CD133-cells were isolated from F6 tumor cells and their purity were detected by the flow cytometric analysis. F6-CD133+and F6-CD133-cells were injected subcutaneously into the right or left back of the same nude mouse to determine their tumorigenic capacity respectively.To determine their self-renewal and multi-linkage differentiation capacity,the proper culture conditions for F6 CSCs were investigated from the three kinds of medium:the completed serum culture medium(10%FBS DMEM),the low serum culture medium(2%FBS DMEM),the serum-free culture medium supplemented with LIF,EGF and bFGF.Proliferation and differentiation of F6-CD133+and F6-CD133-cells were analyzed by cell growth curve,clone formation and Western blot.In addition,the sensitivity of F6-CD133+and F6-CD133-cells to cisplatin(DDP) and 5-fluorouracil(5-FU) were tested by MTT assay.Microarray was used to search for different expression genes.The results of microarray were confirmed by Real-time quantitative PCR.DAPK and FHIT gene promoter methylation was detected by the methylation specific PCR(MSP) in FMSC and F6 cells. After treated with 5-Aza-CdR,flow cytometry and hoechest 33342 staining were used to detect DNA content and apoptosis.The expression of DAPK and FHIT gene was detected by RT-PCR.Results The percentage of CD133~+ cells in F6 tumor cells was 21.1%.After sorting,the purity of F6-CD 133+and F6-CD 133-cells were 86%and 97.9%,respectively.Tumor formation in nude mice showed that F6-CD133+cells were more tumorigenic than F6-CD133-cells,only 1×10~3 F6-CD133+cells could form the tumor.Cell growth curve,clone formation and AKT,ERK protein expression of F6-CD 133+cells showed that F6-CD133+cells possesed stronger proliferation potential than F6-CD133-cells.The F6 CSCs could proliferate and keep the properties of CSCs in low serum culture condition DMEM supplemented with 2% FBS.F6-CD133+cells possessed multi-linkage differentiation capacity in complete serum culture condition DMEM supplemented with 10%FBS. Antitumor drugs assay confirmed F6-CD133+cells were more sensitive to DDP and more resistant to 5-FU than F6-CD133-cells.Gene expression profile based on microarray analysis revealed that the number of different expression genes beween F6 and FMSC was 4715, up-regulated genes was 2911,down-regulated genes was 1804,beween F6-CD133+and F6-CD133-cells was 673,up-regulated genes was 520, down-regulated genes was 153.The different expression genes were identified by Real-time quantitative PCR.Promoter hypermethylation of DAPK and FHIT genes existed in F6 cell line,the expression of FHIT gene was appeared in F6 cells with 5-Aza-CdR treatment.Conclusions Cancer stem cells from F6 cell line which is mutated from FMSC are successfully isolated using CD133-PE antibody by FCM. Our findings suggest that F6-CD133+cells possess the basic characteristcs of cancer stem cells which possese stronger potential of proliferation and differentiation.There are so many different expression genes between F6-CD133+and F6-CD133-,F6 and FMSC.These genes were involved in many cell signal passways such as MAPK,Wnt,Cell cycle.They are different sensitivity to DDP and 5-FU.In addition,FHIT gene hypermethylation might be one of molecular events involved in the development and transformation of FMSC into F6 tumor. |
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