Effects Of Umbilical Cord Mesenchymal Stell Cells On The Biological Behavior Of U87Malignant Glioma Cells

Objective:This experiments based on the established vitro culture and identification by the UMSCs, with UMSCs and U87MG cells co-cultured in vitro, simulate the microenvironment of glioma growth. Investigate the method and the expected results by UMSCs and U87MG cells directly or indirectly co-culture, observed cell morphology, cell cycle and ultrastructural changes by U87MG cells, to analysis the biological behavior of U87MG cell by UMSCs. Summarize UMSCs and U87MG cell co-culture methods and experiences, approaches and provide the basis for further research.Methods:We used fresh full-term umbilical cord separation of the tissue culture method and trypsin digestion under sterile conditions, and trypsin digestion separation DMEM containing10%FBS (fetal bovine serum, FBS) in5%CO2incubatorcultivation, the conventional medium change, passaged processing, to take P4generation UMSCs using flow cytometry to identify UMSCs surface markers CD29, CD44, HLA-ABC. Take P4generation UMSCs,and concentration formulated into1x106cell/ml suspension,and with1x106cells/ml U87MG that continuous and stable passage for more than3generations,of a single cell suspension co-cultured directly or indirectly, to be divided into direct co-culture group, UMSCs supernatant dope indirect co-culture group, UMSCs supernatant1:2indirect co-culture group, UMSCs supernatant1:4indirect co-culture group and UMSCs supernatant1:8indirect co-culture group. We Observation cell morphology each group with optical microscope, Determined OD values before and afte co-culture by MTT assay.and chart the growth curve. Collect of U87MG cells and measured growth cycle by flow cytometry and ultrastructure observed. Results:(1) Identification of Markers of UMSCs Surfaces by flow cytometryDigested UMSCs with trypsin separation and passed generation and culture in vitro, make UMSCs constantly obtain purified. Expression of the surface markers CD29. CD44. HLA-ABC, and other ratios are more than90%.of the P4UMSCs. Having the typical characteristics of stem cells(2) Determination of cell viability results bymultiply-table tournamentMTT assay indicated inhibition U87MG with UMSCs. The U87MG cells with1:8,1:4,1:2and undiluted UMSCs supernatant after96hours and the inhibition rate is17%,24%,37.2%and46.4%, respectively, and has a time and concentration-dependent manner. Significant difference between the control group (U87MG cells with normal DMEM culture group).(3) The U87MG cells morphological changes after U87MG cells co-cultured with UMSCsCultured for48hours in a normal application DMEM observation cell morphology of U87MG cells with inverted phase contrast microscope. U87MG cells were adherent growth, cells clear and polygonal, were nested growth, Cytoskeleton disappeared, integrated into the film, some cells off the cell body shrinkage deformation after join UMSCs supernatant cultured for48hours. With prolonged incubation time.the cells morphology from polygonal to spindle-shaped, protrusions disappeared and the rupture of the cell skeleton structure.(4) The UMSCs morphological changes after U87MG cells co-cultured with UMSCsUMSCs primary cells adherent growth medium was changed after2times DMEM culture flasks bottom of the cell extensional. Deformation into polygonal or fusiform, refraction, nuclei were larger. The cells were chaged by single cell layer to nested distribution after culture2weeks later, digest the cells were passaged and cultured for12hours, the cells gradually changes from round to spindle, or polygonal morphological rules, strong refraction. Continue to foster See markedly accelerated cell proliferation,72-96hours can be covered with about90%of the bottom of the bottle. The majority of UMSCs cytoskeleton disappeared, protrusions disappear and local cobblestone proliferation co-cultured with U87MG cells and MUSCsfor24hours.(5) U87MG cells cell cycle under UMSCs supernatantRoled in end of the UMSCs supernatant concentration of1/2and1/4after12h,24h and48h. Used FCM determination of the cell cycle:have hypodiploid peak (sub-G1) with1/2UMSCs supernatant cultured the U87MG cells12h and accounting for0.43%of the total number of cells. Training with UMSCs supernatant dope U87MG cells after48h. significant sub-G1. accounting for16.97%of the total number of cells.1/2UMSCs supernatant1/2UMSCs supernatant culture U87MG cells allows in S phase arrest after12h. The UMSCs significantly alter the U87MG cell cycle and cycle arrest (S phase arrest).(6) Apoptosis in U87MG cells co-cultured with UMSCsMeasured total cultured the U87MG cell apoptosis after further by flow cytometry, With the co-culture time extend and UMSCs of supernatant concentration increase, the U87MG cell apoptosis showed a clear upward trend, showing significant differences for the time and concentration(p<0.01).Conclusion:After morphological changes by co-culture before and after U87MG UMSCs of co-culture, the observation of the growth curve and the effect of the growth cycle analysis to draw UMSCs supernatant U87MG inhibition, and showed atime-and concentration-dependent manner.(1) UMSCs can significantly reduce the adhesion between the U87MG cells;(2) UMSCs could significantly inhibit the proliferation of U87MG cells;(3) U87MG cells apoptosis rate with the time and UMSCs supernatant concentration increased after UMSCs co-cultured with U87MG cells(4) The U87MG nuclear condensation and fragmentation, mitochondria and other organelles showed vacuolar degeneration and apoptosis change after UMSCs co-cultured with U87MG cells.