OY-TES-1: Effect On Biological Behaviors Of Mscs

Aim: To understand the effect of OY-TES-1 on biological behaviors of MSCs.Methods: (1) Discontinuous density gradient centrifugation was used to separate MSCs from bone marrow, then MSCs were cultured with 10% FBScontaining L-DMEM medium and were passaged by using trypsin combined with EDTA to obtain a single stem cell populations; the following methods were performed for identification of MSCs: microscopic observation of cell morphology, detection of cell growth by CCK8 assay, flow cytometric detection of cell surface molecular markers (CD 105, CD90, CD44, CD29, CD45, CD 14 and HLA-DR), and induction of MSCs to osteoblasts and adipocytes with conditional medium; MSCs were stored by using 5% DMSO, then we compared cell morphology, survival rate, growth condition and surface molecular marker expression between pre-freezing and post-resuscitation cells in order to determine the effect of cryopreservation.(2) RT-PCR, immunocytochemistry, immunofluorescence and Western blot were used to detect the expression of mRNA and protein of OY-TES-1 gene,respectively; OY-TES-1 siRNA was transfected by liposome, RT-PCR and Western blot were used to determine the optimal transfected time-point and the interference efficiency of OY-TES-1 in MSCs; after down-regulation of OY-TES-1, morphological characteristics and surface molecular markers expression of MSCs were observed.(3) Cell proliferation was detected by CCK8 assay, flow cytometry was used to detect the cell cycle and apoptosis; Western blot was performed to detect cycle associated protein Cyclin E and apoptosis related protein Caspase-3;scratch repair experiment and Transwell migration assays were performed for the detection of cell migration ability.Result: (1) By discontinuous density gradient centrifugation, we obtained MSCs from bone marrow in vitro, MSCs were well purified through culture and continuous passage, showing typical morphologic features, and were uniformly positive for CD105 (98.65%), CD90 (99.5%), CD44 (99.48%) and CD29(99.16%), nearly negative for CD45, CD14 and HLA-DR, and exhibited multilineage differentiation potential. MSCs were well cryopreserved by using 5% DMSO, and after resuscitation, their morphological characteristics, survival rate and cell growth ability did not change significantly when compared with pre-freezing cells, and still expressed series of MSCs related surface molecular markers (CD 105, CD90, CD44 and CD29).(2) Not only OY-TES-1 mRNA, but also OY-TES-1 protein can be found in MSCs, and OY-TES-1 protein located in both cytoplasm and nuclear of MSCs.The down-regulation of OY-TES-1 in MSCs did not affect the morphological features and related surface molecular marker expression of MSCs.(3) After down-regulation of OY-TES-1 gene in MSCs, cell proliferation decreased, cells in G1 phase increased, but S and G2 phase cells decreased with inhibiton of Cyclin E expression, the percentage of apoptotic cells increased with enhanced expression of Caspase-3, as well as cell migration ability attenuated. These results suggested that the down-regulation of OY-TES-1 in MSCs, can influence cell proliferation, cell cycle distribution, apoptosis and migration ability in MSCs, and OY-TES-1 may be involved in MSCs biological behaviors, but the exact mechanism is not clear.Conclusion: (1) The expression of OY-TES-1 in MSCs was determined; by using RNAi technology, OY-TES-1 in MSCs can be effectively down-regulated.(2) OY-TES-1 was involved in biological behaviors of MSCs, such as cell proliferation, cell cycle, apoptosis and migration ability.