Experimental Of Enrichment Liver Cancer Stem-like Cells Based On Tumor Resistance Characterics

Objecitve:Establish the model enrichment of liver cancer stem cells with resistance characteristics of cancer stem cell, both in vivo and in vitro experiments were conducted to increase the proportion of liver stem cell by giving increasing dose of ADM.The aim of this study is to solve the "bottleneck" effect in cancer stem cell culture, and increase the proportion of cancer stem cell.Methods:Selecting human liver BEL-7404cell lines as the research object, in vitro and in vivo with the chemotherapy drug intervention as the basic method, set up enrichment mode of liver cancer-like stem cells. The main intervention models are listed as follows:(1) in vitro liver cancer stem cells enrichment mode, long-term chemotherapy drugs intervention model,gradually increasing chemotherapy drugs (adriamycin) concentration, duration8months,②in vitro short-term chemotherapy drug intervention model:different concentration doxorubicin intervention liver cancer cells, after the short and long term chemotherapy drugs duration4days, join in to the stem cell culture medium cultivate environment.(2) In vivo liver cancer stem cells enrichment mode:inoculate liver cancer cells in BALB/C-nu mice subcutaneous tumor animal model, the adriamycin (8mg/kg) and cyclophosphamide (100mg/kg) intervention, when the diameter of tumor is reach1.5cm, using primary cultured,consecutively passaging BEL-7404for2-3times in BALB/C-nu miceThe index of liver cancer stem cell enrichment model was tested as follow:(1)the single-cell cloning formation experiment,(2) the flat cloning formation experiment,(3)"gloable culture"namely the medium formed balloon stem cells (4) CXCR4flow cytometry technique.Results:(1) In vitro liver cancer stem cells enrichment model:The BEL-7404cells in vitro cloning proliferation ability is strong, single-cell cloning formation rate, flat cloning formation rate is respectively(21.60±6.22)%and(161.25±18.30)%o,BEL-7404doxorubicin short-term intervention cells BEL-7404/ADM-S single-cell cloning formation rate, flat cloning formation rate is respectively (3.13±0.75)and(24.33±6.11)%o.(2) Long term chemotherapy drug intervention model:Using adriamycin in vitro,low concentration for incremental method can be induced drug-resistant liver cancer cell lines formed BEL-7404/ADM(0.4μg/ml),multiple resistance is292times. BEL-7404/ADM-L cells can discharge ADM out of the cell,but single-cell cloning formation rate, flat clonining formation rate lower than parental cells, it is respectively(0.02±0.01)and(113.75±18.89)%o.BEL-7404, and BEL-7404/ADM-S, BEL-7404/ADM-L cells in culture medium CXCR4under standard of expression rate respectively is (4.33±2.07)%,(0.73±0.33)%,(5.25±2.76)%.Under the stem cell culture medium the expression rate of CXCR4is respectively (23.84±6.90)%,(35.59±8.39)%,(28.41±15.61)%. BEL-7404, and BEL-7404/ADM-S, BEL-7404/ADM-L in low adhesion hole on board with stem cells are not find the "balloon".(3) In vivo liver cancer stem cells enrichment mode:Inoculated BEL-7404or BEL-7404/ADM liver cancer cells in BALB/C-nu mice. Enrichment of BEL-7404or BEL-7404/ADM-L liver cancer cells was performed by consecutively passaging BEL-7404for2-3times in BALB/C-nu mice with doxorubicin(ADM), cyclophosphamide(CTX) respectively, and the tumor formation rate was100%.Inoculate parent cells forming xenotransplantation tumor group:the adriamycin of intervention BEL-7404P1, BEL-7404P2cells single-celled cloning formation rate respectively is:(16.0±1.41)%,(73.80±5.66)%.BEL-7404P1, BEL-7404P2flat cloning formation rate is respectively (31.00±14.14)%o,(98.00±16.97)%o.After cyclophosphamide of intervention BEL-7404P1, BEL-7404P2, BEL-7404P3single cells cloned formation rate respectively is:(42.76±15.89)%,(42.13±3.38)%,(39.23±14.35)%.BEL-7404P1, BEL-7404P2, BEL-7404P3flat cells cloned formation rate respectively is:(266.50±6.40)‰,(478.00±18.06)‰,(557.25±13.10)%‰。 Inoculate drug-resistant cells of xenotransplantation tumor group:the intervention of adriamycin BEL-7404/ADM P1, BEL-7404/ADM P2single cells cloned formation rate respectively is (31.00±14.14)%,(98.00±16.97)%,BEL-7404/ADM P1, BEL-7404/ADM P2flat cells cloned formation rate respectively is (178.75±56.91)‰,(906.50±59.38)%o.After intervention of cyclophosphamide, BEL-7404/ADM P1, BEL-7404/ADM P2, BEL-7404/ADM P3single cells cloned formation rate respectively is:(22.00±3.58)%,(33.22±13.66)%,(47.05±6.93)%.BEL-7404/ADM PI, BEL-7404/ADM P2, BEL-7404/ADM P3flat cells cloned formation rate respectively is (480.50±28.11)%o,(452.63±92.68)%o,(415.00±19.30)%o. Inoculate parent cells of xenotransplantation tumor group:the intervention of adriamycin BEL-7404P1, BEL-7404P2cells CXCR4expression rate respectively is:(1.46±1.75)%,(0.50±0.09)%, After intervention of cyclophosphamide BEL-7404P1, BEL-7404P2, BEL-7404P3single cells cloned formation rate respectively is:(2.78±3.35)%,(3.78±3.26)%,(2.06±0.77)%.Inoculate drug-resistant cells of xenotransplantation tumor group:the intervention of adriamycin BEL-7404/ADM P1, BEL-7404/ADM P2single cells cloned formation rate respectively is:(2.94±3.34)%,(15.90±23.24)%.After intervention of cyclophosphamide BEL-7404/ADM P1, BEL-7404/ADM P2, BEL-7404/ADM P3single cells cloned formation rate respectively is (2.05±1.70)%,(24.38±0.35)%,(6.55±2.20)%, After the intervention of adriamycin BEL-7404/ADM P2cell suspension ball formation rate is (11.27±4.43)%。Conclusion:Human liver BEL-7404cells appeare the part of characteristics of the cancer stem cells.By the adriamycin intervention drug-resistance tumor cell lines in BALB/C-nu mice subcutaneous inoculate transplantation, can enrichment of characteristics of the liver cancer-like stem cells. Through the test and analysis, CXCR4is not suit the new liver cancer stem cells positive marker.