Preliminary Study On The Sorting,identification And Source Of Lymphatic Endothelial Cells

Objective:1.Preliminary study on the purification and basic biological characteri-stics of tumor lymphatic endothelial cells.2.To investigate the possibility of CD133~+stem cell-like cells differentiation into tumor lymphatic endothelial cells in tumors.Methods:1.The model of colon cancer(HT29)or non-small cell lung cancer(A549y)was established in BALB/c Nude nude mice.The existence of lymphatic vessels in the tumor was verified by immunohistochemistry and immunofluorescence.Then the endolymphatic endothelial cells were isolated by anti-Podoplanin and anti-LYVE-1 immunomagnetic beads.Flow cytometry and immunofluorescence methods were used to verify the purity of sorting,and tube forming experiments were used to verify the angiogenic characteristics of tumor endolymphatic endothelial cells isolated by anti-Podoplanin and anti-LYVE-1immunomagnetic beads.2.The tumor model of non-small cell lung cancer(A549y)with fluorescently labeled was constructed.Stem cell-like tumor cells were isolated from A549y tumor by anti-CD133~+immunomagnetism beads,and their dryness was identified by flow cytometry,WB and globular characteristics.Cell scratch test and Transwell invasion test were used to explore the effects of different cytokines on the migration and invasion of stem cell-like tumor cells selected by CD133~+immunomagnetic beads.Then the cytokines which had the greatest influence on the invasion and migration ability were selected to induce and culture CD133~+stem cell-like tumor cells.Immunofluorescence,flow cytometry,q-PCR and Western blot were used to detect whether the cells were induced to express lymphatic endothelial markers.Results:1.Lymphatic endothelial cells were found in tumors by immunohistochemistry and immunofluorescence.Lymphatic endothelial cells were sorted from tumor tissues using anti-Podoplanin and anti-LYVE-1immunomagnetic beads.The results of flow cytometry showed that the positive cells Podoplanin and LYVE-1 were more than 80%after sorting by anti-Podoplanin and anti-LYVE-1 immunomagnetic beads sorting kits.Immunofluorescence results showed that the cells obtained after sorting by anti-Podoplanin and anti-LYVE-1 immunomagnetic beads sorting kits expressed Podoplanin and LYVE-1.Matrigel tube-forming experiments showed that the cells obtained after sorting by anti-Podoplanin and anti-LYVE-1immunomagnetic beads sorting kits have good tube forming ability,indicating that they have endothelial cell biological characteristics.2.CD133~+tumor cells were sorted from tumor tissues using anti-CD133~+immunomagnetic beads.The results of flow cytometry showed that the positive cell CD133 expression rate was over 90%after sorting by anti-CD133~+immunomagnetic beads sorting kit.The results of Western blot showed that the cells obtained after sorting by anti-CD133~+immunomagnetic beads sorting kit expressed the stem cell marker Nanog protein.Microsphere formation experiments showed that cells obtained by sorting with anti-CD133~+immunomagnetic beads have stem cell globular properties.The cytokines VEGF-C,b FGF and CXCL12,which are closely related to the invasion and migration ability of CD133~+tumor cells,were added to the endothelial medium to form the induction medium.After the positive cells were induced by induction medium for 12 days,the positive cells were induced by anti-CD133~+immunomagnetic beads sorting kit for 12 days.Immunofluorescence detection showed that all the induced cells expressed Podoplanin and LYVE-1.The expressions of PROX-1 and VEGFR-3 were detected by fluorescence quantitative PCR.Western blot results showed that the induced cells expressed PROX-1 and VEGFR-3.The endothelial cell phagocytosis test showed that the endothelial cell phagocytosis had the biological characteristics of endothelial cell phagocytosis.The Matrigel tubule formation test showed that the induced cells could form lumen-like structure,indicating that the cells induced by induction medium had tube-forming characteristics.Conclusion:1.The lymphatic endothelial cells were successfully isolated from the tumor,and the selected cells expressed lymphatic markers and had the characteristics of tubule formation.2.Stem cell-like tumor cells were successfully isolated from the tumor and identified.It was found that the migration and invasion ability of CD133~+cells under the combined action of VEGF-C,b FGF and CXCL12 was stronger than that under the condition of single factor.At the same time,some CD133~+stem cell-like tumor cells could be induced to differentiate into lymphatic endothelial cells by adding these factors.Using the model of fluorescent labeled non-small cell lung cancer(A549y),it was verified that lymphatic endothelial-like cells originated from some stem cell-like cells in tumor cells,which provided a new strategy for inhibiting tumor growth and metastasis.