Research Interaction Of CMA And Macroautophagy Through Inhibition And Activation CMA In EPCs

Objective: To analyze autophagy-related protein LC3and Lamp-2a ofmacroautophagy and CMA when CMA was blockage by SB203580and activated by6-ANin EPCs, then research the relationship between macroautophagy and CMA.Methods:1.Rat bone marrow was obtained by rinsing bone marrow cavity，then bonemarrow mononuclearcells were isolated by gradient centrifugation using Ficollcentrifugate. mononuclearcells were then cultivated with EGM-2MV medium cultivation.MTT assay were used to draw growth curve of cells．Cultvated cells werc identified byimmunofluorescence、angiogenesis and immunohistochemistry.2.EPCs were culturedthrough adding different concentrations of SB203580（2.5uM、5.0uM、10uM、20uM、40uM）and6-AN（2.5mM、5.0mM、10mM、20mM、40mM）for24hours. Observe theproliferation ability assay by MTT; Flow cytometry(FCM) was used to detect cellapoptosis; Western blot detection of LC3-II、Lamp-2a expression changes. Observechanges of Lamp-2a incytoplasm by laser scanning confocal microscope. SPSS17.0software was used for analysis. Mean expression by （x±s）,single-factor analysis ofvariance used to compare the average of each group.（P＜0.05） with statisticalsignificance.（P＞0.05）without statistical significance.Result:1.Rat bone marrow EPCs were isolated successfully by ficoll centrifugate invitro.2. The number of EPCs decreased with the concentration(2.5uM、5.0uM、10uM、20uM、40uM)of the SB203580, while the number increased with theconcentration(2.5mM、5.0mM、10mM、20mM、40mM) of6-AN, compared with the controlgroup was statistically significant(P＜0.05).3.6-AN(2.5mM、5.0mM、10mM、20mM、40mM)groups can decrease apoptosis of EPCs(P＜0.05), but increased apoptosis at theconcentration of40mM. While SB2036580(2.5uM、5.0uM、10uM、20uM、40uM)groupscan increase apoptosis of EPCs(P＜0.05).4. SB2036580(2.5uM、5.0uM、10uM、20uM、 40uM)groups can increase the expression of LC3-II in EPCs by detection of western blot,which induce the expression of Lamp-2a;6-AN (2.5mM、5.0mM、10mM、20mM)groupscan increase the expression of Lamp-2a by western blot, and induce the expression ofLC3-II.6-AN groups significantly increased the expression of Lamp-2a by Laser scanningconfocal.Conclusion: Inhibition of CMA can up-regulate macroautophagy of EPCs andpromote apoptosis by SB203580; activation of CMA can inhibit macroautophagy andpromote the proliferation of EPCs. Our work support a direct cross-talk between CMA andmacroautophagy. One autophagy pathway will up-regulate when the other autophagypathway was blockage. There is a compensatory mechanism between the autophagicpathways of CMA and macroautophagy.