The Effect Of Rapamycin On Endothelial Progenitor Cells

Objective: In vitro,we induce differentiation of endothelial progenitor cells (endothelial progenitor cells, EPCs), and then apply the activator of autophagy–rapamycin to intervent to explore the biological characteristics of EPCs after intervention, for such as deep vein thrombosis treatment to provid new ideas and experiments.Method: 1. Density gradient centrifugation Wistar rat bone marrow mononuclear cells (bone marrow-derived mononuclear cells, BMMNCs), endothelial progenitor cell medium (EGM-2MV) induced to differentiate into EPCs, and then adherent cells were divided into five groups, one for the control group (normal culture group), and the remaining 4 groups were added with rapamycin 0.01,0.1,1,10μg / L of EGM-2MV medium in each group , cultured for 24,48 h. 2. observe the proliferation assay by MTT. 3. monodansylcadaverine (MDC) staining for detection of autophagic vesicles .4. EPCs transmission electron microscope to view ultrastructure . 5.Western blot detection of LC3 protein expression .6 . flow cytometry for cell cycle progression and apoptosis. SPSS17.0 software used for analysis.Results: 1, EGM-2MV culture medium isolated by Ficoll bone marrow mononuclear cells can be induced to EPCs; 2. The effect of rapamycin was measured by the OD value,it was significantly decreased in a dose and time dependent ,the control group and treatment group differed significantly (P <0.05). 3. Autophagic vesicles were stained by MDC successfully and we can observed more intuitive the presence of autophagic vesicles and the fluorescence intensity with time and with increasing concentration. 4. By electron microscopy we observed such as mitochondrial swelling, autophagic body double membrane structure, and chromatin margination ultrastructure changes. 5. LC3-Ⅱprotein expression of cells was significantly increased in the treatment group, significantly higher than control (P <0.05) 6. Observed cells showed time and concentration with the increasing trends; drugs make cells were arrested in the G1/G0 phase of and S phase, thus the G2 / M phase of mitotic cells were clearly shown a downward trend.Conclusion: Rapamycin can significantly affect cell cycle progression and inhibit cell value, and promote apoptosis and autophagy.