Research On The Mechanism Of Bone Mesenchymal Stem Cells Promote Endothelial Progenitor Cells Proliferation

Objective:To explore a method for simultaneously isolate,culture and identify murine Mesenchymal Stem Cells and Endothelial Progenitor Cells from bone marrow of C57BL/6, And to study the effect of MSCs on promoting EPCs proliferation in co-culture system without contacting.Methods:1. MSCs were isolated by the whole bone marrow adherent method from C57BL/6 mice bone marrow; to identify the adherent cells, which of the third passage were used to assay cell surface markers by flow cytometry. To assess the differentiation capacity of MSCs, osteogenic and chondrogenic inductions were performed in vitro.2. EPCs were isolated by anchorage velocity-dependent separation method. The third passage of EPCs was used to assay cell surface markers by flow cytometry. And the cultured cells were further identified by tube formation experiment in vitro.3. The MSCs and EPCs of third passage,were seeded into Transwell System by 1:1.The nether chamber were EPCs and the upper chamber were MSCs as an experimental group. At the same time only set the same density of simple EPCs seeded in the nether chamber as the control group. MTT assay and BrdU fluorescence assay were used to detect the proliferation of EPCs which influenced by MSCs.Results:1. Obtained the MSCs and EPCs successfully from bone marrow cavity of C57BL/6J mice. MSCs were immunopositive for surface markers of mesenchymal, but were negative for the hematopoietic markers.EPCs expressed surface markers of haematopoietic stem cell, and they could also expressed endotheliocyte marker.MSCs and EPCs were also Identified from the differentiation potential:Adipogenic,Osteogenic and chondrogenic differentiation results showed that the cells could differentiate into fat,chondrocytes and osteoblasts when cultured in induction medium;EPCs could form tube-like structure on Matrigel.2. MTT method demonstrated a higher OD value in experimental group at 24,48,72,96h, compared with control group,the difference was statistically significant (P<0.05).3. BrdU fluorescence labeling showed that compared with the control group, the proportion of BrdU positive cells in the experimental group significantly increased (P<0.05).Conclusion:The method of modify differential adhesion can simultaneously isolate,purify and amplify the MSCs and EPCs from mouse bone marrow.The MSCs can promote the EPCs proliferation in co-culture system without contacting.Objective:To discuss the effect of IGF-1 and downstream IGF-1R during the process of EPCs proliferation stimulated by MSCs.Methods:1. ELISA was used to detect the expression of IGF-1 in the MSCs and EPCs.2. IGF-1 in different concentration level was applied to stimulate EPCs and tested its proliferation3. IGF-1-siRNA and IGF-1R-siRNA were respectively used to intervene the IGF-1 expression by MSCs and IGF-1R expression by EPCs. Besides, IGF-1mRNA and IGF-1RmRNA expression level were examined by RT-PCR4.Using MTT method tested EPC proliferation level in each group.Results:1. The expression level of IGF-1 in MSCs culture fluid is much higher than that in EPCs and such differenceis significant (P<0.01).2. The number of EPCs and OD value increased significantly, after stimulating EPCs by IGF-1 in different concentration, when IGF-1 came to 20,50,100 and 200 ng/mg, especially in 100ng/ml (P<0.05).3. Both IGF-1mRNA and IGF-1RmRNA expression level detected by RT-PCR decreased, after the expression of IGF-1 in MSCs was intervened by IGF-1-siRNA and the expression of IGF-1 R in EPCs by IGF-1R-siRNA (P<0.05).4. By using MTT method to detect EPCs proliferation level, compared with the control, EPCs proliferation in experimental group was significantly inhibited after IGF-1-siRNA intervened MSCs (P<0.05); similar phenomenon appeared after IGF-1R-siRNA intervened EPCs (P<0.05).Moreover, IGF-1 neutralizing antibody can inhibit EPCs proliferation during its promotion by MSCs (P<0.05).Conclusion:IGF-1 is able to upgrade EPCs proliferation level, possibly in the way of combining with the downstream receptor.Objective:To reveal whether the PI3K/Akt signalling pathway is involved in EPCs proliferation induced by MSCs.Method:Three groups were established:1. EPC group; 2. EPC+IGF-1 group; 3. EPC+IGF-1+LY294002 group. LY294002 is a protein kinase inhibitor that is able to block Phosphotidylinsitol-3-Kinase cell signal pathway; MTT method was applied to examine EPCs proliferation level in three different groups; RT-PCR was used to detect mRNA expression of IGF-1 receptor and Western-blot was employed to test phospho-Akt protein expression. Cells in EPCs group and EPCs+IGF-1R siRNA group were transfected with GFP and subsequently transplanted into C57BL/6 mice. Moreover,EPC proliferation was observed under fluorescence microscope on mice model with femoral artery injury. Based on the above-mentioned, we try to research the probable mechanisms of EPCs proliferation influenced by IGF-1.Results:MTT results demonstrated that LY294002 inhibited EPCs proliferation under the stimulation of IGF-1. In the two group added with IGF-1, RT-PCR suggested IGF-1 receptor mRNA expression level is significantly higher than that in the first group (p<0.05); Wester-blot results indicated the usage of LY294002, the specific PI3K inhibitor, can significantly downregulated Akt protein phosphorylation level. Fluorescence microscope revealed the significantly higher EPC expression level in EPCs group, compared with EPCs+IGF-1R siRNA group.Conclusion:IGF-1 can stimulate EPC proliferation probably by activating PI3k/Akt signalling pathway.