| Objective:At present the incidence of liver cancer ranked fifth in the global,which is one of the global high-risk sexual malignant tumor,at the top of the incidence in guangxi region in China.At this stage,the main treatment are surgical resection,radiotherapy and chemotherapy,which are the conventional traditional methods.For the past few years,new concept of tumor treatment For the past few years,putting forward new concept of tumor treatment,is due to cancer stem cell theory which is widely used in a variety of parenchymal cells in vitro and animal experiment in vivo.A growing number of evidences point out the viewpoint of tumor heterogeneity.It says that cancer stem cells are derived from A tiny amount of extremely vigorous growth cells which possess the ability of self-renewal,infinite proliferation potential,differentiation and high tumorigenicity.Such cells played an extremely important role on tumor occurrence and development process,aspect of tumor recurrence after treatment,rehabilitation of drug resistance.In cancer cells,but the other most of the cells do not have infinite proliferation potential,after almost follows the brief matured,finally for the development of the process of death.We think there is such a group of liver cancer cells of liver cancer stem cells,Our research cultivated liver cancer stem cells,in serum-free medium.Explore the potential influence of PI3K/AKT signaling on the stem cell-like characteristics of a CD90+ subpopulation of human hepatocellular carcinoma cell line MHCC-97H.Methods:1.Use complete medium and no related growth factor serum containing medium cultivated liver cancer cell line MHCC-97H,respectively make its adherent and suspension into sphere-forming cells,for subsequent experiments.2.We detected spherical cell sources by Glycogen staining reagent.3.Adherent cells and sphere-cells are separately in the cell cycle of the state.4.Flow cytometry tested the content of CD90+cells in adherent cells and sphere-forming cells,by Streaming antibody marker CD90 + cells.5.Two groups of Cell proliferation rate determined by MTT detection,which is CD90+subsets and CD90-subgroup,after treating with PI3K/AKT pathway inhibitor LY294002.6.After the treatment of CD90+ subsets by PI3K/AKT pathway inhibitor(LY294002),we use flow cytometry to analyze the change of CD90+ stem cells in MHCC-97H cell line.7.it is Liver cancer stem cells the expression of related genes CD90,NANOG,SHP2 quantity changes,in CD90+subsets and CD90-subgroup,after treating with LY294002.8.Western blot test their associated protein expression.9.Tablet clone formation experiment testing CD90 + subsets and CD90-subgroup,the effects of cloning,treating with LY294002.10.It is Comparing CD90+subsets and CD90-subgroup to nude mice subcutaneously into tumor ability,in different grade concentration,and the change of forming tumors ability with LY294002.11.Compare CD90+the group of cells and CD90-the group of cells in nude mice after subcutaneous tumor in the liver cancer stem cells related antibody levels;Compare two subgroup cells respectively after the use of PI3K/AKT pathway inhibitor,the expression of liver cancer stem cells related antibodies in control group and treatment groupResults:1.Liver cancer cell MHCC-97H lines can be stable passages in the completely medium during 5 days.In serum free medium,suspensing tosphere-forming cells also canbe stable passages about 7 to 9 days.2.Glycogen dyeing shows that is cancer of the liver experiment the sphere-cells sources.3.Sphere-cells of cell cycle are in a relatively static state,also similar as the characteristics of stem cells.4.Flow cytometry tested the content of CD90+cells in liver cancer stem cells,as follow:the expression of sphere-forming cells>the expression of adherent cells(P<0.05).5.The proliferation rate of CD90+subsets>the proliferation rate CD90-subgroup,by MTT.6.Human HCC steam cells are existed in MHCC-97H cells.The treatment of LY294002 caused the decline in the proportion of human HCC stem cells,in CD90+subsets,by Flow cytometry.(P<0.05).7.The expression quantity of Liver cancer stem cells related gene(CD90、NANOG、SHP2)significantly decreased after adding inhibitor LY294002 in CD90+subsets group(P<0.05);so does not change significantly CD90-subgroup expression quantity.8.The related expression quantity of Western blot(CD90、SHP2)of Liver cancer stem cells significantly decreased after adding inhibitor LY294002 in CD90+subsets group(P<0.05).The expression of P-AKT which is PI3K/AKT signaling pathways downstream of specific protein is decreased.(P<0.05)9.LY294002 obviously reduced the clone ability of CD90+ subpopulation,control group and experimental group(96.13±3.88)%和(60.82±7.03)%,P<0.01。but there is no significant effect for CD90-subgroup’s clone ability,control group and experimental group(22.54±4.47)%和(19.62±3.21)%,P=0.41.10.The ability of tumor-formation in CD90 + subgroup cell were significantly stronger than CD90-subgroup,with the same number of cells.11.LY294002 is able to reduce the tumorigenic ability for CD90+subgroup,but not for CD90-subgroup.12.The expression of CD90+subgroup protein with no drug was obviously stronger than the expression of CD90-subgroup protein with LY294002 by CD90 or SHP2,but not for CD90-subgroup.Conclusion:Special serum-free medium can better enrichment of liver cancer stem cells,CD90+ cells constitute a subpopulation of cancer stem cells among MHCC-97H cells,and their stem cell-like characteristics depend on PI3K/AKT signaling.These results lay the groundwork for further study of stem cells in HCC onset and progression,and they suggest possible therapeutic targets. |
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