The Empirical Study Of Effect Of Autophagy Inhibition On Endothelial Progenitor Cells

Objective: To investigate the effect of proliferation,apoptosis and cell cycle of autophagy inhibition on endothelial progenitor cells.thereby to create a new method for the therapy of the deep venous thrombosis.Methods:1,Bone marrow-derived mononuclear cells were isolated from rat bone marrow by ficoll and cultured with EGM-2MV medium cultivation.observe the features and biological characteristics of EPCs. 2,Experimental design for the five groups, the control group and four concentrations of 3-methlyadenine (3-MA). 1.25mmol/l,2.5mmol/l,5mmol/l,10mmol/l. MTT method used to detect various concentrations (3-MA) proliferation of endothelial progenitor cells. 3,Flow cytometry(FCM) were used to detect cell apoptosis and cell cycle. 4,Monodansylcadaverine staining and Transmission Electron Microscope (TEM) were used to examine autophagy induced by 3-MA in endothelial progenitor cells.5,Western blot analysis were used to detect the LC3-II protein expression changes.6 SPSS 13.0software was used for analysis. mean expression by (±s). Single-factor analysis of variance used to compare the average of each group. (P <0.05) have statistically significant.(P﹥0.05) without statistical significance.Result:1.The rat bone marrow EPCs were islated successfully in vitro.2. 5mmol/l 3-MA promote proliferation of endothelial progenitor cells. compared with the control group was statistically significant (P <0.05). 10mmol/l 3-MA inhibit the proliferation of endothelial progenitor cells . Compared with the control group was statistically significant (P <0.05). The remaining two groups had no significant effect on proliferation of endothelial progenitor cells. 3. 10mmol/l 3-MA can promote apoptosis of endothelial progenitor cells(P <0.05). EPCs in the other groups have no significant difference in apoptosis rate. 5mmol/l and 10mmol/l 3-MA influence endothelial progenitor cell cycle distribution. 10mmol/l 3-MA can occur for S phase of endothelial progenitor cells increased (P <0.05). G2/M phase cells decreased (P <0.05).5mmol/l 3-MA can reduce the G0/G1 phase cells and increased S and G2/M phase cells(P <0.05). There were no significant differences in the other two groups on the cell cycle. 4. Autophagy inhibition was induced in EPCs at the concentration of 5mmol/l and 10mmol/l 3-MA as detected by both MDC staining and TEM. 5. 3-MA can reduce LC3-II protein expression of endothelial progenitor cells by detection of Western blot . 5mmol/l and 10mmol/l 3-MA significantly reduced protein expression of endothelial progenitor cells(P <0.05).Conclusion: 3-MA could inhibit endothelial progenitor cells in autophagy. A moderate inhibition of autophagy can promote the proliferation of endothelial progenitor cells. Excessive autophagy inhibition can inhibit the proliferation of endothelial progenitor cells. Redistribution of the cell cycle may be one of the mechanisms affecting cell proliferation. Moderate inhibition of autophagy did not significantly affect the apoptosis of endothelial progenitor cells. Excessive inhibition of autophagy may promote apoptosis of endothelial progenitor cells.