Preliminary Analysis Of Side Population Phenotype In Human Gastric Cancer Cell Lines

Objective:In cancer stem cell theory,it is considered that the cancer tissues are in the same manner with normal tissues,which are composed of cells with diversity differentiation potency.There are a small portion of special tumor cells acting as stem cells in tumor tissues,which have unlimited proliferation and differentiation potential. These cells are tumor initiating cells and they play an important role in tumorigenesis,development,recurrence and metastasis.Recently,the cancer stem cells have becoming the hot spot in cancer researching domain.The fundamental problem of researching cancer stem cells is how to isolate,purify,culture and identify the cancer stem cells from cancer tissues or cells.Lately,a new method for enriching stem cells is applying by more and more investigators.The cells isolated by this method are called side population cells,which can efflux DNA binding fluorescent dye,Hoechst33342,so they demonstrate low fluorescence after be excited by the UV light.The SP cells have been discovered from various tissues of human and animals, furthermore they also exist in many kinds of solid tumors and cancer cell lines.In addition to this,they are shown to have stem cell characteristics and enrich the stem cell population.The objective of this experiment is to identify whether there are side population cells in human gastric cancer cell lines BGC-823 and MGC-803,moreover observe the morph of SP cells.We also want to know the relationship between SP phenotype and stem cell marker ABCG2 and other ABC transport proteins ABCA3,ABCB1.Methods:1,Dye the BGC-823 and MGC-803 cells cultured in culture flask with Hoechst33342 for 90 minutes and then examine them by fluorescence microscope, observe if there are SP cells and the morph of them,moreover count the percentage of the SP cells.2,Extract the total cell RNA and use polymerase chain reaction to detect the expression of ABCA3,ABCB1,ABCG2 in both BGC-823 and MGC-803 cell lines.3,Mount the BGC-823 cells on the cover slip and stain them with Hoechst33342 for 90 minutes,then fix the cancer cells with 4%paraformaldehyde;subsequently stain the cells with the mouse anti-human ABCG2 monoclonal antibody or goat anti-human ABCA1 polyclonal antibody,and then stain them by fluorescence antibody goat anti-mouse or rabbit anti-goat which labeled by FITC.Observe them with fluorescence microscope.Results:1,SP phenotype has been detected in both BGC-823 and MGC-803 cell lines.The SP cells are small and occupied 1.8%and 1.4%of total cells in BGC-823 and MGC-803 cell lines respectively.2,The total RNA has a good purity and quantity.Result of RT-PCR revealed that ABCA3 and ABCG2 were positive in both BGC-823 and MGC-803 cell lines,but the expression of ABCB1 was not apparent.3,Immunofluorescece chemical stain confirmed the expression of ABCG2 in the BGC-823 cells,and the SP cells expressed ABCG2 phenotype.Conclusion:1,There are side population cells in human gastric cancer cell lines BGC-823 and MGC-803,and the SP phenotype accounts for about 1～2%of all tumor cells.2,The ABCG2 and ABCA3 transporters are both expressed in these two human gastric cancer cell lines. 3,The SP cells express stem cell marker ABCG2.