| Background:More and more experiments showed that tumor was composed of non-uniform tumor cells, tumor cells possessed heterogeneity. Even in a tumor or a tumor cell line, tumor cells are different in growth velocity, proliferation, differentiation, invasion, drug resistance. Subpopu-lation clones are different in cell phenotype and differentiation.Tumor heterogeneity is also different in tumorigenicity, according to different tumorigenicity of the immune deficiency mice, tumor cells can be divided into high tumorigenic ability of cancer stem cells (tumor stem cells, TSCs) and low tumorigenicity or non-tumorigenic ability of tumor cells (non-tumorigenic cancer cells).Although few as the cancer stem cells are,they can self-renew, proliferate and differentiate, they are the source of tumor occurrence, development, recurrence, invasion and metastasis.Present studies showed that cancer stem cells exist in a variety of hematologic malignancies and solid tumors.Cancer stem cells and non-tumor cells are different in tumor proliferation and differentiation capacity.In vitro culture conditions,colony formation is significantly different and shows different subpopulation clones heterogeneity.It was reported that glioma, skin cancer, breast cancer and other subpopulation clones had morphological heterogeneity, one of which had strong ability to develop tumors and contained cancer stem cells. Therefore, cell clone heterogeneity,especially the heterogeneity of morphology may help to detect and sort tumor stem cells. There is evidence of some liver cancer or liver cancer cell line has heterogeneity,and may be related to the presence of liver cancer stem cells (liver cancer stem cells, LCSCs).Whether human hepatoma cell line HepG2 exists clonal heterogeneity or not? Whether the heterogeneity of subpopulation clones can be used to select liver cancer stem cell from HepG2 cell line is still not reported. The mechanism of using cloning morphological heterogeneity to identify and separate tumor stem cells is not clear. So the research of relationship between cloning heterogeneity of hepatocellular carcinoma and liver cancer stem cell have an important theoretical and practical signi-ficance.Objectives:1.To study the differences of human hepatoma cell line HepG2 cell subpopulation in clonal morphology heterogeneity and cell cycle.2.To investigate human hepatoma cell line HepG2 with different cloning cells of liver cancer stem cells markers CD133, ABCG2 and CD90 staining.Methods:1. Using finite dilution to culture human hepatoma cell line, HepG2 cells were cultured single-cell cloning,different clones were observed the morphology under inverted microscope.2.Using flow cytometry to detect the cell cycle of cell subpopulation cloning.3.Using immunohistochemistry to detect cell subpopulation cloning of liver cancer stem cell marker CD133, ABCG and CD90 expression.4.Western blotting was used to detect different subpopulation of the ABCG2 protein expression of cloning.Results:1. Human hepatoma cell line HepG2 of subpopulation existed different cloning in appearance:Clone A was multangular and tight in shape. While clone B showed a sharp and loose feature.2. Human hepatoma cell line HepG2 clone A and clone B the cell cycle of results:clone A:G0/G1 phase (72.13±3.25)%, S phase (17.26±0.93)%, G2/M phase (10.11±0.32)%; clone B:Go/G, phase (50.63±1.26)%, S phase (25.07±1.24)%, G2/M phase (24.31±0.14)%. clone A of the G0/G1 phase was significantly higher than clone B, there was significant difference between the two groups (P<0.05). 3. The positive expression rates of liver cancer stem cell marker CD133,ABCG2 and CD90 in hepatoma cell clone A and clone B were 66.67%(6/9),71.12%(5/7),80%(4/5) and 10%(1/10),15.38% (2/13),9.09%(1/11) respectively,there was significant difference between the two groups (P<0.05)4.Western bloting test showed that the clone A relative molecular mass of 70000 Da ABCG2 protein expression occurred around target band, as comparing with clone B, clone A of ABCG2 protein expression was significantly increased comparing with the control group, there was signi-ficant difference between the two groups (P<0.05).Conclusions:1. Heterogeneity of tumor cell clones exists in human hepatoma cell line HepG2, which may arise from Liver cancer stem cell differentiation.2. Different cell subpopulation clone in human hepatoma cell line HepG2 may be comprised of liver cancer stem cells.
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