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Background: Liver cancer is one of the diseases that serious threat to human health .in recent years, cancer stem cell theory proposed for liver cancer research points to new direction. Theory of cancer stem cells thank that cancer stem cells is the seed of the tumor cells and the original cells of tumor Occurrence, development and maintenance , but also the underlying causes of tumor recurrence and metastasis. Current treatment methods of liver cancer is surgery, However, most of HCC cases are presented at an advanced stage and no longer operable with these surgical treatments, it is the result of the existence of cancer stem cells, only the removal of these cells that can to cure cancer.However, the liver stem cell research is still at a preliminary stage, how to get a lot of liver cancer stem cell is difficult, because the number of liver cancer stem cells are extremely limited both in the peripheral blood or liver tissue. The separation of liver cancer stem cells has important significance on our study.Researchers generally use the Methods of FACS or MACS to identify the liver cancer stem cells , because they express CD90 and CD44 but there are many deficiencies.Zeng yi xin had first proposed the new benefits: not only the cancer stem cells derived from malignant transformation of adult cells, but also from ordinary tumor cells in chemotherapy stimulation induced genomic instability is generated at Xiangshan meeting in 2004. On this basis, Yang et al use chemotherapy successfully enriched breast cancer stem cells in 2007;Levina et al cultured human lung cancer cells use to chemotherapy and successfully enriched the lung cancer stem cells in 2008 . Many studies have shown that CD90 is recognized the surface marker of the liver CSCs cell , Yang, etc. found in many and various stem cell-related surface markers, expression of CD90 cells are related to the potential Tumorigenicity of the liver, we continue to study these cells and found these cells also expressed CD44, CD44 is a cell surface hyaluronan receptor, and play an important role in the invasion and metastasis of liver cancer. Therefore, this study was to establish a method ,the method is to enrich, amplify, identify a cell subsets with the characteristics of liver stem cell ,it provide a new method for further study a new treatment of liver cancer.Objective:1. In vitro simulation chemotherapy in body, explore the conditions for chemotherapy and get drug surviving cells .2.Explore the morphological differences and the expression of CD90, CD44 between the liver cancer cells and drug surviving cells and to analyze the significance.Methods:1.Obtained drug surviving cells by in vitro chemotherapy, compared the changes in cell morphology before and after chemotherapyobserved under inverted microscope.2.Using immunocytochemical staining method to detect the expression of cell surface markers CD90, CD44 before and after chemotherapy.3.Using flow cytometry to detect the expression of cell surface markers CD90, CD44 before and after chemotherapy.Results: 1.The morphological changes between liver cancer cells and drug surviving cells:before chemotherapy: We observed a single uniform round cells under the microscope before adherent ,they adherent within 24 hours, and then began to split proliferation, cell growth was the convergence , closely arranged, clearly outlines ,connected pieces, various shapes, it includ spindle, polygonal, polygonal;after chemotherapy:Cells decreased significantly, loosely arranged and shrant into small balls.2.Flow cytometry Results: control group labeled①,Diluted 100 times labeled①, Diluted 50 times labeled③, Diluted 30 times labeled③, Diluted 25 times labeled③, Diluted 20 times labeled⑥.(1)Comparison of percentage of CD90 between liver cancer cells and drug surviving cells:The percentage of CD90 ~+ cells expressed in the six group were (0.74±0.1)%, (8.54±0.68)%, (14.96±0.55)%, (22.16±0.37)%, (21.1±0.21)%, (19.04±0.16)%. The group’s F value is 4.240, (P < 0.01), the difference has statistically significant. The①compared with other group the P less than 0.01, the difference has statistically significant. And with the reduction of dilution the percentage of CD90 ~+ cells is increased, when the concentration of drug increased a certain degree, even if you increased the concentration the percentage of CD90 ~+ cells is declined.(2)Comparison of percentage of CD44 between liver cancer cells and drug surviving cells:The percentage of CD44 ~+ cells expressed in the six group were (0.25±0.08)%, (4.98±0.23)%, (9.06±0.25)%, (13.39±0.32)%, (13.10±0.33)%, (12.46±0.20)%. The group’s F value is 4.50, (P < 0.01), the difference has statistically significant. The①compared with other group the P less than 0.01, the difference has statistically significant. And with the reduction of dilution the percentage of CD44 ~+ cells is increased, when the concentration of drug increased a certain degree, even if you increased the concentration the percentage of CD44 ~+ cells is declined.3. Results of immunofluorescence staining:(1)Comparison of percentage of CD90 between liver cancer cells and drug surviving cells:The expression of cells fluorescent particles is 1% in Control group, , the number of fluorescent particles cells expressed 23% in chemotherapy residual group ,the difference was statistically significant.(X~2=22.917,P<0.05).(2) Comparison of percentage of CD44 between liver cancer cells and drug surviving cells:The expression of cells fluorescent particles is 2% in Control group, , the number of fluorescent particles cells expressed 14% in chemotherapy residual group ,the difference was statistically significant.(X~2=9.783,P<0.05).Conclusion:1.CD90, CD44 expression was significantly increased after HepG2 liver cancer cell lines were treatment by chemotherapy in vitro, suggesting that HCC cells under pressure of chemotherapy could enrich liver cancer stem cells.2.With the increase of the concentration of chemotherapeutic agents, expression of CD90, CD44 percentage increase, when added to a certain concentration, continued to increase concentration, the expression of CD90, CD44 percentage has decreased, when the peak concentration in plasma concentrations of 1 / 30 times the expression is the highest percentage.
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