Establishment The Co-culture Cell System Of Human Ovarian Carcinoma Cell With Directional Highly Lymphatic Metastasis-endothelial Cells And Study Their Biological Characteristics

Part Ⅰ IntroductionOvarian cancer is the gynecologic malignancy in the incidence of third place in the mortality rate ranks, but NO.1in the malignant disease. As the ovarian cancer in pelvic anatomy on the hidden depths of the early onset of the lack of specific symptoms and signs, difficulty in early detection and diagnosis, about60%-70%of ovarian cancer when patients are diagnosed advanced stage. In order to investigate the ovarian tumor microenvironment which affected the ovarian cancer directional lymphatic metastasis, we applied co-culture system to study biological characteristics of human ovarian carcinoma cell with directional highly lymphatic metastasis (SKOV3-PM4), human umbilical venous endothelial cells (HUVEC) and human lymphatic endothelial cells (HLEC). We collected current literature and made a brief overview of human ovarian carcinoma cell with directional highly lymphatic metastasis and its tumor microenvironment. Part II Establishment the co-culture cell system of Human ovarian carcinoma cell with directional highly lymphatic metastasis-Human umbilical venous endothelial cell and study their biological characteristicsObjective:To establish the interaction system between SKOV3-PM4and HUVEC cells and to study their biological characteristics.Methods:The cells of SKOV3-PM4and HUVEC were labeled with green and red fluorescent respectively. To collect the culture medium of SKOV3-PM4cells as SKOV3-PM4condition medium and the culture medium of HUEVC as HUEVC condition medium, The co-culture cell system and condition medium cuture system were established. The growth curve of the cells was determined by MTT assay. The ultra-structure and morphological changes of cells in the condition medium cultivation and co-culture system were observed by hematoxylin and eosin staining and electron microscope. The change of each cells biological characteristics were detected by laser scanning confocal microscope, flow cytometry, healing assay, Trans-well assay, gelatin zymography and so on.Results:Compared with the single culture SKOV3-PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division. The cell ratio of G1phase decreased and cell ratio of G2and S phase increased. The cell of SKOV3-PM4in HUVEC condition medium grows more quickly than SKOV3-PM4in normal medium, the results of healing assay and trans-well assay showed that invasiveness and migration capability enhanced. Compared with the single culture HUVEC, the cells which cultured in SKOV3-PM4condition medium show the morphological significantly changed and vacuolization occurred in the cytoplasm. The cell ratio of G1phase increased and cell ratio of G2and S phase decreased. The HUVEC cell of in SKOV3-PM4condition medium grows same as HUEVC in normal medium.The result of healing assay and transwell assay showed that invasiveness and migration capability enhanced. Two kinds of cells became fusion in the co-culture system under the laser scanning confocal microscope. Gelatin zymography assay showed that MMP-2low-expressed in SKOV3-PM4supernatant and didn’t expressed in HUVEC supernatant and high-expressed in SKOV3-PM4-HUVEC co-culture supernatant.Conclusions:The biological characteristics of SKOV3-PM4and HUVEC show significantly changes after co-culture and condition medium of culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway. Part III Establishment the co-culture cell system of Human ovarian carcinoma cell with directional highly lymphatic metastasis-Human lymphatic endothelial cell and study their biological characteristicsObjective:To establish the interaction system between SKOV3-PM4and HLEC cells and to study their biological characteristics.Methods:The cells of SKOV3-PM4were labeled with green fluorescent protein. The cells of HLEC were labeled red fluorescent respectively. To collect the culture medium of SKOV3-PM4cells as SKOV3-PM4condition medium and the culture medium of HLEC as HLEC condition medium. Co-culture cell system and condition medium of culture system were established. The growth curve of the cells was determined by MTT assay. The ultra-structure and morphological changes of cells in the condition medium cultivation and co-culture system were observed by hematoxylin and eosin staining and electron microscope. The change of each cells biological characteristics were detected by laser scanning confocal microscope, flow cytometry. healing assay, Trans-well assay, tube formation assay, gelatin zymography and so on.Results:Compared with the single culture SKOV3-PM4, the cells which cultured in HLEC condition medium showed the increase of pseudopodia and nuclear division. The cell of SKOV3-PM4in HLEC condition medium grows more quickly than SKOV3-PM4in normal medium. The results of healing assay and trans-well assay showed that invasiveness and migration capability enhanced. Compared with the single culture HLEC, the cells which cultured in SKOV3-PM4condition medium show the morphological significantly changed and vacuolization occurred in the cytoplasm. The HLEC cell of in SKOV3-PM4condition medium grows quickly than HLEC in normal medium. The results of healing assay, transwell assay and tube formation assay showed that invasiveness, migration and canaliculization capability enhanced. Two kinds of cells became fusion in the co-culture system by the laser scanning confocal microscope. Gelatin zymography assay showed that MMP-2low-expressed in SKOV3-PM4supernatant and didn’t expressed in HLEC supernatant and high-expressed in SKOV3-PM4-HLEC co-culture supernatant.Conclusions:The biological characteristics of SKOV3-PM4and HLEC show significantly changes after co-culture and condition medium of culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway. Part Ⅳ Initial research of screening for the differentially secreted proteins in human ovarian carcinoma cell with directional highly lymphatic metastasis by two-dimensional gel electrophoresis and mass spectrometryObjective:To analysis the different secreted protein between the SKOV3-PM4and SKOV3cells and to study their function in tumorous invasion.Methods:To collect of the culture supernatant of SKOV3and SKOV3-PM4. The proteins exprerssed in human ovarian carcinoma cell with directional highly lymphatic metastasis were analyzed by two-dimensional gel electrophoresis. The differentially expressed proteins in SKOV3-PM4were also analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry.Results:Proteins were separated by two-dimensional gels.There were selected about twenty differentially expressed proteins in human ovarian carcinoma cell with directional highly lymphatic metastasis. Seven proteins were successfully identified by MS. in which five proteins were overexpressed in SKOV3-PM4and the other two proteins were underexpressed.Conclusions:There are distinct secreted proteins in SKOV3and SKOV3-PM4cells. The different proteins are related to tumorous invasion process.