| To detect the effects of CD133-downrultation on the radiosensitivity of CD133+HepG2cancer stem cells (CSCs) in hepatocelluar carcinoma.Part1Sorting of CD133+-HepG2cells and checking of their stemness properties.Methods:MACS was used to isolate CD133+and CD133-cells from HepG2cell line. Flow cytometry was used to detect the expression of CD133before and after cells isolation. Spheres-forming assay in vitro and the NOD/SCID mice transplantation tumor experiments in vivo were performed to validate the cancer stem-like properties of sorted CD133+cells. Colony forming assay was used to compare the colony-formation ability between CD133+and CD133-cells.Results:CD133+and CD133-cells were successfully sorted by MACS, and1.36±0.20%and87.62±1.92%CD133+cells were detected by flow cytometry before and after isolation, respectively. Spheres-forming assay showed that CD133+cells could form spheres and in serum-free culture media with grow factors, whereas the CD133-cells showed absent failed to stay alive in the serum-free culture media. NOD/SCID mice tumorigenicity assay showed that just1,000CD133+cells could form subcutaneous xenografts5weeks after inoculation, whereas10,000CD133-cells could not form tumors in the same condition. Hematoxylin and eosin (H&E) stating analysis showed a highly cellular mass and clear nucleis below the CD133+cells injection site. Colony forming assay showed that CD133+cells have greater ability to form clones than CD133-cells, which had a higher cloning efficiency (35.03±2.35)%compared to CD133-cells’ cloning efficiency (6.4±0.72)%(P<0.01).Conclusion:The results revealed that CD133+HepG2cells demonstrated higher tumor spheres formation ability, colony forming ability and tumorigenesis capacity than CD133-cells, which could be considered as CSC-like subsets of liver cancer cells.Part2Infection of CD133+HepG2by lentivirus with CD133sequenceMethods:Targeted silence towards CD133gene was performed and experimental groups were divided into blank control group, negative-transfection group and positive-tranfection group. The efficiency of infection was observed by inverted fluorescence microscope. RT-PCR and Western blot were used to detect the gene and protein expressions of CD133. Colony formation assay was applied to detect the proliferative ability of cells.Results:The expression of green fluorescence protein were detected under inverted fluorescence microscope3days after infection, which peaked on the fifth day. The infection rate was more than80%. The expression level of CD133in mRNA and protein level was significantly decreased in positive-transfection group(P<0.01). Colony formation assay showed that the proliferation ability of CD133+liver cancer stem cells was decreased after CD133-downregulation(P<0.01).Conclusion:The results showed that lentivirus-mediated shRNA could down-regulate the expression of CD133in the CD133+liver cancer stem cells, and inhibit their proliferative ability.Part3Detection of radiosensitivity by colony formation assay and exploration of mechanismsMethods:Colony formation assay was applied to detect colony formation efficiency and survival rate after irradiation with different doses; survival curve was drawn and radiobiology parameters were counted. Flow cytometry was used to test cell cycle and apoptosis rate.Results:The dose survival curve of positive-transfected group was down overall than the other two groups. The values of radiobiology parameters PE, SF, SF2, Do, Dq and N were decreased obviously compared to negative-transfection group and blank control group. The SER (sensitive enhancement ratio) was1.37±0.02(P<0.01). The results of flow cytometry and apoptosis assay showed that S phase cells decreased in posi tive--transfection group whereas G2phase cells and apoptosis rate increased significantly (P<0.05).Conclusion:After CD133-downregulation, the radiosensitivity was obviously increased. The change of cell cycle and the increasement of apoptosis rate may be the mechanisms to enhance radiosensitivity. |
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