Research Of Isolation Culture And Separation Of Mouse Bone Marrow Lymphatic Endothelial Progenitor Cells

Objective On the basis of mouse bone marrow endothelial progenitor cells (EPCs),lymphatic endothelial progenitor cells (LEPCs) were isolated,and the intervention of exogenous vascular endothelial growth factor-c (VEGF-C) was introduced to improve the experimental method of in vitro isolation,culture and induction differentiation of lymphatic endothelial progenitor cells (LEPCs).Methods 1.In this study,the endothelial progenitor cells (EPCs) from bone marrow cells were extracted by modified differential adhesion method in C57BL/6 mice,and cells not adhered to the wall within 48 h were collected and cultured in EGM-2MV complete medium until the third generation.2.The third-generation cell surface marker CD34 CD133 VEGFR-3 was detected by flow cytometry,and the percentages of VEGFR-3+CD34+cells and VEGFR-3 +CD133+ cells in EPCs were analyzed 3.LEPCs cells with positive VEGFR-3 surface markers were selected by flow cytometry,and the expression of CD34 and CD133 on the surface were identified by immunofluorescence chemistry.4.The LEPCs sorted by flow cytometry were cultured to the third generation,and the proliferation ability of LEPCs was detected by MTT colorimetry.5.LEPCs were cultured and induced to differentiate by introducing exogenous vascular endothelial growth factor-c (VEGF-C).Morphological changes of LEPCs were observed under light microscope.After 21 days of induced differentiation,expression of lymphatic endothelial hyaluronic acid receptor-1(LYVE-1)was detected by immunofluorescence chemistry.6.LEPCs were induced and differentiated by VEGF-C at different concentrations in groups of 50ng/ml,100ng/ml and 150ng/ml,and the expression of LYVE-1 in the three groups was detected by immunofluorescence chemistry.Results 1.Flow cytometry showed that VEGFR-3+CD34+cells accounted for 0.67% of EPCs cells,and VEGFR-3+CD133+cells accounted for 1.52% of EPCs cells.Immunofluorescence chemical assay showed that CD34 and CD133 were expressed on the cell membrane of VEGFR-3+cells by flow cytometry.2.The proliferation of LEPCs was stronger in vitro.There was no significant change in the number of cells in the first 3 days.On the 4th day,the logarithmic growth phase began.After the 11 th day,the plateau was in the plateau.The growth curve showed an "S" shape.3.LEPCs were differentiated by VEGF-C induction,and LYVE-1,a special marker of lymphatic endothelium,was detected on its surface by immunofluorescence.4.VEGF-C with a concentration of 100ng/ml was an ideal concentration for bone marrow LEPCs in mice induced differentiation in vitro.Conclusion LEPCs can be isolated and purified by differential adhesion method and flow cytometry.LEPCs can be amplified by EGM-2MV medium.VEGF-C can induce LEPCs to differentiate into lymphatic endothelial cells.