| The thesis includes two parts:Part 1. Expression of CD133 correlates with differentiation of human colon cancer cellsIn this study, we tested the CD 133 expression in 44 widely used human cancer cell lines. Interestingly, five of 8 CD133 positive cell lines were colon cancer cells. We sorted the CD133high/+ and CD133- subpopulation of HCT116 cells and found that the CD133high/+ HCT116 cells grew quicker (in vivo) and generated more colonies after 14 days incubation. CD133high/+ HCT116 cells had higher percentages of cells in S and G2/M phases than their CD133- counterparts. In ongoing xenograft tumor formation experiments in BALB/c-nu/nu mice, we did not find a difference in tumorigenic potential of CD133high/+ versus CD133-subpopulations. Cultivation of both CD133high/+ and CD133-HCT116 cells resulted in a redistribution of antigen expression to its original proportion. Our results suggest other markers rather than CD 133 alone should be used to enrich for colon CSCs, especially in cultured colon cancer cell lines.Next we explored the relationship between CD133 expression and differentiation. HT29 cells were sorted 4 scales according to CD133 staining, our results suggested that differentiation status (ALP activity) was inversely correlated with CD 133/2 expression in HT29 cells. Then, we found that HT29 and HCT116 lose their CD133/1 and CD133/2 reactivity (flow cytometry) in a time-and dose-dependent manner after differentiation induced by SB, but the total protein level (Western Blotting) in the cell did not change. We also synthesized three pairs of siRNAs, two of which could efficiently reduce CD133 expression in vitro. There was no appreciable effect on the proliferation, cell cycle distribution, colony formation and differentiation of HCT116 and HT-29 cells by knocking down the expression of CD133. CD133 seems not to be a prerequisite gene for cell growth and differentiation.In all, CD 133 may be more a differentiation indicator rather than a specific stem cell lineage marker in colon cancer cell lines.Part 2. Establishment of tumor metastasis models expressing green-fluorescent proteinFour cell lines were transfected with the plasmid pEGFP-N1, and limited dilution was employed to screen four monoclonal cell strains expressing GFP:human cervical adenocarcinoma cell HeLa-GFP, human colon carcinoma cell HCT116-GFP, human mammary adenocarcinoma cell MDA-MB-231-GFP and murine cervical carcinoma cell U14-GFP.8×106 HeLa-GFP cells were transplanted into BALB/c-nu/nu mice. The latent period of tumor mass formation was 3-5 days and its tumorigenicity is 100%. The tumor growth of HeLa-GFP was well defined by the Photometries. Only one mouse was shown to harbor lymphatic metastasis by Photometrics 60 days after transplantation.8×106 U14-GFP cells were transplanted into C57BL/6J mice, the latent period of tumor formation was 2-4 days and its tumorigenicity is also 100%. The metastasis process of U14-GFP was depicted through the observation by Photometrics on 22,27,37 and 52 days post-transplantation. The incidence of pulmonary metastasis and lymphatic metastasis of U14-GFP was 67% and 100% respectively when the tumor volume was> 5 cm3. We also transplanted HCT116-GFP subcutaneously and injected them into tail vein of BALB/c-nu/nu mice. The HCT116-GFP tumor stably expressed green flourcence in vivo by Berthold Viviperception Fluorescence Imagining System. Fourty three days after HCT116-GFP tail vein injection, GFP positive metastasis tumors were detected by Berthold.The expression of CD44 and E-cadherin were checked in HeLa-GFP and U14-GFP tumor tissues, CD44 was positive and E-cadherin was negative in both tumors by immunohistochemistry.In conclusion, we successfully established four monoclonal tumor cell strains stably expressing GFP. Transplantation of these cells into mice can establish tumor metastasis models which could be used for future visualized tumor research in vivo.
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