Screening And Identification Of Biological Characteristics Of Liver Cancer Monoclonal Cell

Aim :1.Screening of monoclonal cell lines from six successful primary tumor cells using Incu Cyte ZOOM system.2.Two monoclonal cells were selected to detect the clonogenic ability using Incu Cyte ZOOM system.3.Three monoclonal cells derived from the same primary tumor cells were selected and identified for their morphological,cell cycle,growth,migration and invasion ability,stem cell markers and other biological characteristics.Methods :1.Single cell cloning and clonogenic ability of tumor cells were analyzed by real-time dynamic tracking and full-hole imaging with Incu Cyte ZOOM system.2.The cell morphology of the monoclonal cell line was observed by fluorescence inverted microscope,and HE staining for calculating nuclear mass ratio using IPP.3.Using Incu Cyte Zoo M to compare cell growth,migration,invasion ability of monoclonal cell lines.4.Detecting HBV DNA loading in cell supernatants of monoclonal cell lines by fluorescence quantitative PCR.5.Detecting the expression of AFP,CK18,CK19,GPC3,Hep Par-1,PCNA and Vimentin in monoclonal cell lines were detected by immunofluorescence technology.6.Detecting the expression of DLK-1,CD47,CD13,CD133,CD24,CD44,CD90 and Ep CAM and cell cycle of monoclonal cell lines in the surface markers of stem cells by flow cytometry.Results: From the successful cultivation of six primary tumor cells(78T,83 N,92N,216 N,216T1,216T2),we screened 89 continuous proliferation of monoclonal from 5 strains,of which 67 clones were amplified and cryopreserved;18 monoelone cell lines were not continuous proliferation and 28 polyclonal cells consisting of two or more cells were excluded by the Sequence Diagram.The analysis for clonogenic ability of two monoclonal strains(216T1-SC-B11,216T2-SC-H11)showed that the clonogenic rate of plate method(35.17%,13.17%)was significantly higher than that of Incu Cyte ZOOM(23.13%,5.51%)at 14 days,and the difference was statistically significant(P < 0.05);The colony formation rate is(35.63%,13.22%)at full aperture imaging when extending to 21 d,and there is no statistical difference compared with the plate method.The morphology of 92N-SC-C12 and 92N-SC-F4 were similar,and the 92N-SC-B2 was different from that of the other two cells.Using the Incu Cyte ZOOM system,the growth capacity of monoclonal strains was analyzed by cell fusion model in phase difference mode.The doubling time of the monoclonal strain 92N-SC-F4 was less than that of the other two monoclonal cells,the doubling time(h)was 35.00 ± 5.00,and the doubling time of the three monoclones was less than 92N(60.67 ± 2.08);By comparing the migration and invasion ability of three monoclonal cells in the wound width model,we find the 92N-SC-F4 was stronger than that of the other two strains.Cell cycle analysis showed that the G2 phase ratio of 92N-SC-F4 was larger than the other two monoclones,and the G1 phase was smaller than the other two clones.Detecting the HBV DNA loading in the supernatant of the cells,both the three monoclonal and primary tumor cells was positive,and the number of copies was > 105;Immunofluorescence results showed that CK18 was positive in monoclonal cell lines and primary tumor cells,and the protein Hep Par-1 is negative;3 strains of monoclonal cells did not express AFP;protein Vimentin,GPC3,PCNA weak expression;CK19 expression differences.The expression of protein Vimentin,GPC3 and CK18 in the cytoplasm of four cells and the expression of PCNA protein were found in the nucleus.The results of stem cell marker analysis showed that CD13 and CD47 were highly expressed in monoclonal and 92 N cells,and the expression ratio was more than 99%.The expression of DLK-1,CD24,CD90 is low or not;The expression of CD44 in monoclonal strain ratio(> 82%)is higher than primary tumor cells 92 N(76.7%);The expression of CD133 was different,the monoclonal strain 92N-SC-F4(41.5%)was higher than that of the other three strains;The expression ratio of Ep CAM in monoclonal strain 92N-SC-B2 and 92N-SC-F4 was much higher than that of monoclonal strain 92N-SC-C12(49.90%).Both 4 cells were positive for CD47 / CD13/Ep CAM/CD44/CD133,and the expression ratio from high to low in turn is 92N-SC-F4(33.2%)> 92N-SC-B2(26.7%)> 92N(15.6%)> 92N-SC-C12(6.4%).Conclusions: The Incu Cyte ZOOM was applied to screening the single clone and measuring the colony forming efficiency can be simple,accurate,time-saving and effort;The identification of a variety of biological characteristics of monoclonal cells derived from the same individual showed that there is heterogeneity between monoclonal cells;Monoclonal cells differ in the expression of stem cell markers,and also reflect the screening of monoclonal cell lines with tumor stem cell characteristics.