Interaction Of Id1 And E2-2 Modulate The Proliferation Of EPCs

Objective:The incidence rate of coronary heart disease, hypertension and other chronic diseases increased year by year. These artery diseases have hindered the development of society and threatened people’s health. The basic pathological changes of vascular diseases are caused by many kinds of reasons, such as endothelial cell damage and apoptosis, which lead to the formation of vascular stenosis and thrombosis.Therefore, how to realize the re-endothelium of injured vessels and restore the function and structure of blood vessels is currently a hot research topic. In the previous study, it was found that vascular endothelial cell (ECs) can be obtained by proliferation, migration, synthesis and secret a variety of bioactive substances to repair the damaged vascular intima. But ECs is the end-stge of cell cycle and the ability to proliferate and repair the injured vessel is limited. So people become focus on the precursors of endothelial cells, Endothelial progenitor cells(EPCs). By ischemia-hypoxia, cytokines, drugs, injured endothelial cells, EPCs can be mobilizated from bone marrow and spleen, locate the injured vessels and repair the vascular intima by proliferation, differentiation into endothelial cells. Paracrine is another way to repair the injured vessels by EPCs. But how can EPCs proliferate and migrate to the target vessel? The biological mechanism and the factors are still not clear. In the previous studies, our team showed that differentiation inhibitor 1 (Id-1) and transcription factor E2-2 play important roles in the proliferation of EPCs. But the interaction of Idl and E2-2 for EPCs’proliferation still need to research. The aim of this study is to explore the associations between Id-1, E2-2 and which way they regulate and control the proliferation of EPCs.Methods:1.Based on our previous research, density gradient centrifugation was used to isolated and cultured EPCs from rat bone marrow,and EPCs were identified by the function of cells and flow cytometry;2.We bought lentiviral vectors and verified though PCR, enzyme digestion and sequencing. We packaged the lentiviral vectors,verified though titer determination,3.We use immune coprecipitation test to detect the association between Id-1 and E2-2.Over expressed or interfered Id-1 and E2-2 on EPCs, and used CCK 8 method to detect cell proliferation; RT-PCR and Western blotting techniques to detect expression of PI3K, and Akt levels.Results:1.Mononuclear cell from rat bone marrow can be differentiated into EPCs, and these cells have characters of stem cells and ECs; 2.0ver expression and interference lentiviral vector were successfully constructed and can be used used for the following experiment;3.Immune coprecipitation test showed there was protein-protein interaction between Id-1 and E2-2. Compared with the control group, overexpression of Id-1 could decreased the E2-2 expression level, and increased the expression of PI3K/Akt, which lead to cell proliferation; Overexpressing E2-2 reduced the expression of PI3K/Akt and cell proliferation; Common transfection Id-1 and E2-2 suggested that both proteins could influence the expression of PI3K/Akt level together.Conclusion:There was protein-protein interaction between Id-1 and E2-2; Id-1 have positive regulatory role for PI3K/Akt, while E2-2 have a negative regulatory role. The Id-1 and E2-2 could coordinate to regulate the PI3K/Akt signal pathway, thus affecting the proliferation of the EPCs.