Capturing Human Tumor Cells And Detecting Differences Of Cell Surface CD Antigen Expression With Cell Microarray

ObjectiveTumor markers (tumor marker, TM) is produced by the tumor cells and released into the blood, body fluids, reflecting the existence and growth of the tumor. Tumor markers can be divided into the following categories:①cancer embryonic antigen;②enzyme and isozyme;③plasma protein;④cell metabolism;⑤tumor antigen;⑥oncogenes and tumor suppressor gene protein product and trace elements. Tumor antigens are over-expression antigenic material during the cancer development process.Immune response depends on the interaction between cells in the immune system. It directly contact between cells, and the other through the secretion of cytokines or other molecules which mediate the interaction. Function of cell surface molecules includ a variety of cell surface antigens, receptors and other molecules. With identification of monoclonal antibody methods,the same epitope is recognized by monoclonal antibody which come from different laboratories as CD (cluster of differentiation). Some researchers found that normal cells and tumor cells expressed different CD antigens, and CD was related to tumor development.Cell-chip highly detects cell surface CD antigens expression. Sixty-four kinds of monoclonal antibodies fixed on the carrier, and capture cells, basing on specificity of antigen-antibody reactions. Then the cell microarray could be scanned with scanner and analyzed the signals.Cell chip technology can fast analysis the normal and cancer cells surface CD antigens'expression. It is good to clarify the pathogenesis of tumor,and filter the CD phenotypic markers between normal cells and tumor cells.Methods1,Cell culture Normal human gastric cell line GES-1, human gastric cancer cell line 7901, normal human liver epithelial cell line L-02, human hepatoma cell line Bel7404, human pancreatic cancer cell line PANC-1, were small cell lung cancer cell line H446, the NPC cell lung cancer cell line H460, human lung adenocarcinoma cell line A549 and human colon cancer cell line HT29;2,Capturing human tumor cells and detecting differences of cell surface CD antigen expression with cell-chip.3,Verify cells microarray's specificity(1) Validating the CD results of GES-1,7901 and PANC-1 cell surface antigen phenotype with RT-PCR method;(2) Gene sequence of GES-1 and 7901 cell surface expression of CD validat RT-PCR amplification results;(3) Testing the specificity of cell-chip with Cells flow cytometry.4,Screening the fluorescent positive point of cell chips.Results1,CD13, CD29, CD49e, CD55, CD44, CD71, CD95, CD133 antibody points capture the human small cell lung cancer H446 cells; CD9, CD13, CD29, CD44, CD49e, CD55, CD71, CD133, CD95 antibody points capture the H460 cells; CD29, CD44, CD49e, CD55, CD61, CD71, CD133, CD90 antibody points capture A549 cells; CD9, CD15, CD24, CD29, CD44, CD55, CD66, CD71 antibody points capture HT29 cells; CD13, CD29, CD44, CD55, CD71 antibody points capture PANC-1 cells; CD9, CD13, CD15, CD29, CD49e, CD44, CD55, CD71, CD77 antibody points capture Bel7404 cells; CD9, CD13, CD29, CD44, CD49e, CD55, CD56, CD57, CD71 antibody points capture L-02 cells; CD9, CD13, CD15, CD24, CD29, CD44, CD49e, CD55, CD71, CD95 antibody points capture GES-1 cells; CD9, CD13, CD29, CD44, CD49e, CD55, CD71 CD95, CD77, CD133, CD103, CD64 antibody points capture t 7901 cells.2,RT-PCR detect the mRNA of 7901 and GES-1 CD phenotype expression showed that both 7901 and GES-1 express CD9, CD13, CD29, CD44, CD49e, CD55, CD71, CD95, the same with our microarray results; RT-PCR detect the mRNA of PANC-1 CD phenotype expression showed that CD71, CD55 were expressed, the same with our microarray results. 3,Flow cytometry was used to determine 7901 and GES-1 cell surface CD15, CD24, CD44 expression, the same streaming results with our cell microarray4,From FITC-CD14, FITC-CD4, PE-CD3, FITC-CD19, PE-Cy5-CD19, FITC-Cy5-CD2, FITC-CD3, Cy-Chrome-CD22 and PE-CD14, We fond PE-CD13 is the best as cell microarray positive point.Conclusion1,CD29, CD44, CD49e, CD55, CD71, CD133 antibody points capture H446 cells, H460 cells and A549 cells; CD13, CD95antibody points capture H446 cells and H460 cells, did not capture A549 cells; CD9 only capture H460 cells, did not capture H446 cells and A549 cells; CD61, CD90 antibody points only capture A549 cells, did not capture H446 cells and H460 cells.2,CD29, CD44, CD55, CD71 antibody points capture HT29 cells, PANC-1 cells, 7901 cells and Bel7404 cells; CD9 antibody point capture HT29 cells,7901 cells and Bel7404 cells, did not capture PANC-1 cells; CD13 antibody Point capture PANC-1 cells,7901 cells and Bel7404 cells, did not capture HT29 cells; CD15 antibody point capture HT29 cells and Bel7404 cells, did not capture PANC-1 cells and 7901 cells; CD49e, CD77 antibody points capture 7901 cells and Bel7404 cells, did not capture HT29 cells and PANC-1 cells; CD24, CD66 antibody points only capture HT29 cells; CD95, CD133, CD103, CD64 antibody points only capture 7901 cells.3,CD29, CD44, CD55, CD71 antibody points captured H446 cells, the NPC cell lung cancer cell line H460 cells, A549 cells, HT29 cells, PANC-1 cells,7901 cells and Bel7404 cells.4,CD9, CD13, CD29, CD44, CD49e, CD55, CD71 antibody points captured Bel7404 cells and L-02 cells; CD 15, CD77 antibody points only captured Bel7404 cells, did not capture L-02 cells; CD56, CD57 antibody points only captured L-02 cells, did not capture Bel7404 cells.5,CD9, CD13, CD29, CD44, CD49e, CD55, CD71, CD95 antibody points captured GES-1 cells and 7901 cells; CD15, CD24 antibody points only captured GES-1 cells, did not capture 7901 cells; CD77, CD133, CD103, CD64 antibody points only captured 7901 cells, did not capture GES-1 cells; CD15 and CD24 may be GES-1 cell surface markers, CD77, CD133, CD103, CD64 may be 7901 cell surface marker. 6,RT-PCR detected the mRNA of 7901 and GES-1 CD phenotype expression showed that both 7901 and GES-1 expressed CD9, CD13, CD29, CD44, CD49e, CD55, CD71, CD95, the same with our cell microarray; RT-PCR detected the mRNA of PANC-1 CD phenotype expression showed that CD71, CD55 were expressed, the same with our cell microarray.7,Flow cytometry was used to determine 7901 and GES-1 cells surface CD15, CD24, CD44 antigens'expression.The results showed that 7901 cells did not express CD15, CD24,but CD44; GES-1 cell surface express CD15, CD24, CD44, the same with our microarray.8,Determine PE-CD13 is the best as cell microarray positive point.