| Object: In vitro，we induce differentiation of endothelial progenitor cells (endothelialprogenitor cells, EPCs), and then apply the activator of macroautophagy–rapamycin andthe inhibitor of macroautophagy-3-MA to explore intervention EPCs autophagy andmolecular chaperones mediated by the changes in the characteristics of macrophagebiology, for such as deep vein thrombosis treatment to provid new ideas and experiments.Method:1. Bone marrow-derived mononuclear cells were isolated from rat bonemarrow by ficoll and cultured with EGM-2MV medium cultivation.observe the featuresand biological characteristics of EPCs.The cells morphology were observed by microscopy.MTT assay was used to study the activity of proliferation. Also immunocytochemistryperformed to analyze the expression of VEGF-2,vWF and immunofluorescence wereperformed to analyze the expression of CD34and progentitor cell marker CD133dynamically. Using three-dimensional training experiment appraisal EPC whether it hasthe function of forming the blood vessel.2.Adhere cells were divided into two groups, One group of rapamycin.include acontrol group(normal culture group),and the remain six groups were added with rapamycin1μg/L、2.5μg/L、5μg/L、10μg/L、20μg/L、40μg/L of EGM-2MV medium in eachgroup,another group of3-MA have Experimental design for the five groups, the controlgroup and six concentrations of3-methlyadenine (3-MA)1.25mmol/l、2.5mmol/l、5mmol/l、10mmol/l、20mmol/l、40mmol/l. all groups cultured for24h.2.1Experimental design forthe group of rapamycin, the group of3-MA and the control group,MTT method used todetect various concentrations proliferation of endothelial progenitor cells.2.2Western blotanalysis were used to detect the LC3-II protein,LAMP2A protein expression changes.2.3immunofluorescence were performed to analyze the fluorescence intensity of the LAMP2Aprotein.Results:1.the lately-separated bone marrow mononuclear cells(BMMNCs) presentcircular and the size is different.most of the cells start attaching about3days.The morphology formed the shuttle,triangle,the spindle or irregular form.The attached cellswere able to form cells colonies and line up in the core-like structure from5to8days,andget close to coalesce and exhibite the typical cobblestone morphology from8to12days.Cells growth curve was typical “S” shape.EPCs proliferation activity was rapid from6to11days. Cell growth into the platform period after11days. Cell growth curve wastypical “S” shape. The attached cells were positively stained for CD3、VEGFR、vWF、CD133and change dynamically. Early EPCs cell proliferation obviously, but did not forma lumen-like structures.the apparent proliferation of late EPCs to form the networkconnection lumen sample structure.2. The effect of rapamycin was measured by the OD value,it was significantlydecreased in a dose and time dependent,the control group and treatment group differedsignificantly (P <0.05).1.25mmol/l,2.5mmol/l3-MA promote proliferation of endothelialprogenitor cells after24and48hours compared with the control group was not statisticallysignificant (P>0.05).10mmol/l3-MA promote proliferation of endothelial progenitor cellswith the control group was statistically significant (P <0.05).10mmol/l，20mmol/l,40mmol/l3-MA inhibit the proliferation of endothelial progenitor cells. Compared with thecontrol group was statistically significant(P <0.05).Westernblot detected each target protein expression. In the group ofrapamycin,LC3-Ⅱ protein was expressed the trend of increased significantly in1-5ug/lrapamycin concentration range. Intervention group was significantly higher than thecontrol group(p<0.05),but,LC3-Ⅱ protein was expressed the trend of decreasedsignificantly in10-40ug/l rapamycin concentration range With the increasing of theconcentration of the drug. Intervention group of10ug/l、20ug/l rapamycin was higher thanthe control group(p<0.05),but the intervention group of40ug/l rapamycin was lower thanthe control group(p<0.05).LMAP2A protein expression with the increasing of theconcentration of the drug is gradually diminishing expression, significantly lower thancontrol (P <0.05).In the group of3-MA, LC3-Ⅱ p roteinexpression with the increasing ofthe concentration of the drug is gradually diminishing expression, significantly lower thancontrol (P <0.05).LAMP2A protein was expressed the trend of increased significantly in1-10mmol/l3-MA concentration range. Intervention group was significantly higher thanthe control group(p<0.05),but, LAMP2A protein was expressed the trend of decreasedsignificantly in20-40mmol/l3-MA concentration range With the increasing of the concentration of the drug. Intervention group was significantly higher than the controlgroup(p<0.05)The average optical density value of the LAMP2A protein of the rapamycin groupdecreased gradually compared with the control group(P<0.05). The average optical densityvalue of the LAMP2A protein of the3-MA group was higher than the control group in1.25-10mmol/l3-MA range(P<0.05),but the average optical density value of the LAMP2Aprotein of the3-MA group expressed a gradually decline trend in the range of10-40mmol/l3-MA, Compared with the control group were not significant (P>0.05).Conclusion: There is a direct cross-talk relationship between the macroautophagy andmolecular chaperone-mediated autophagy pathway of endothelial progenitor cells... |
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