ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.
ObjectiveTo review the research progress of neural regulation mechanism of vasculogenesis. MethodsThe relevant literature on neural regulation mechanism of vasculogenesis was extensively reviewed. ResultsNeural regulation of vasculogenesis depends on synergistic effect among various cells of neurovascular unit, and co-participation of multiple cytokines, and it is closely related to a variety of repair mechanism, such as nerve regeneration and synaptic plasticity, but the specific mechanism need to be further investigated. ConclusionThe research of the neural regulation mechanism of vasculogenesis will contribute to further understanding repair mechanism of nerves and vessels injuries.
ObjectiveTo explore optimal conditions of isolation, culture and labeled with superparamagnetic iron oxide (SPIO) in vitro of rat bone marrow endothelial progenitor cells, and lay the foundations for the further EPCs tracer study in vivo. MethodsThe EPCs derived from rat bone marrow were isolated and cultured by using density gradient centrifugation, which were labeled with different concentrations SPIO, Prussian blue staining was used to detect the cells labeling rate, MTT assay was used to detect the cells proliferation activity, and Trypan blue staining was used to detect the cells vitality. ResultsEPCs gradually growed in monolayer arrangement about 7 d after cultured. When the concentration of SPIO was 50μg/mL, the highest labeling rate of Prussian blue staining was 90%, the growth state of labeled EPCs were good, and could normal adherent growth and passage. At this time, the cell viability and proliferation activity were the highest through trypan blue staining and MTT assay. ConclusionsEPCs can be labeled with SPIO easily and efficiently when the concentration was 50μg/mL?without interference on the viability and proliferation activity, which lay the foundations for the further EPCs tracer study in vivo.
Objective To determine the transfection efficiency of recombinant adenovirus to endothelial progenitor cells(EPCs) and provide the base of lung cancer therapy by transfecting human herpes simplex virusthymidine kinase（HSV-TK） gene to EPCs. Methods Admove recombinant adenovirus 5F35(AD5F35) which transfected with βgalactosidase(AD5F35LacZ) to the 24 well plate cultivated with EPCs and transfect the EPCs. Stain the EPCs with LacZ kit and calculate the transfection efficiency. Results The blue stain cells were cells transfected successfully with AD5F35LacZ under the optical microscope. The transfection efficiencies of adenovirus to EPCs were different under the premise of the different multiplicity of infection(MOI). In a certain range, the transfection efficiencies rise with the MOI rise. When MOI was 400,the proportion of blue stain cell is the highest, which was 98.38%±1.25%. Conclusion Recombinant adenovirus can transfect EPCs successfully. The transfection efficiencies rise with the MOI rise. When the MOI is 400,the transfection efficiency is the highest.
Objective To study the short and medium term effect of myocardial contractile force by implantation of endothelial progenitor cells （EPCs） in the myocardial infarction model. Methods Hundred and twenty SD rats were equally and randomly divided into experimental group and control group （60 rats in each group）. Acute myocardial infarction model was created by ligation of LAD. Autologous EPCs were purified from peripheral blood then implanted into the acute myocardial infarct site via topical injection. IMDM were used in control group. Specimens and muscle strip were harvested at 3, 6 weeks, 6, 8 and 12 months after EPCs implantation for contractile force study and to detect the expression of vascular endothelial growth factor（VEGF）, basic fibroblast growth factor （bFGF） and Ⅷ factor by immunohistology and video image digital analysis system. Results The expression of VEGF, bFGF and the microvessel counts in experimental group were much higher than those of control group（P〈 0.01） at 3, 6 weeks and 6 months after transplantation. The contractile force in experimental group was better than that in control group（P〈0.01） at the same time. But from 8 months after implantation, the contractile force and so on were not up in the experimental group. Conclusion EPCs, after being implanted into infarct myocardium, shows the ability of improvement of the contractile performance in infarcted myocardium by means of angiogenesis and vasculogenesis and the medium term results are persistent.
The aim of this study was to investigate whether shear stress could promote function of endothelial progenitor cells (EPCs)with Shexiang Baoxin Pill (SBP) treatment in vitro, and to study whether shear stress contributed to vascular injury repair by EPCs. EPCs were isolated and characterized; EPCs' proliferation, migration, adhesion, tube formation and eNOS protein level in vitro were investigated by culturing confluent EPCs in 4 mg/mL SBP under physiological shear stress (15 dyne/cm2) for up to 24 hours. Afterwards, EPCs were transfused into rats after wire-induced carotid artery injury augmented re-endothelialization. The results showed that, compared to the SBP group, the shear stress+SBP group obviously enhanced EPCs proliferation, migration, adhesion, tube formation and eNOS protein expression in vitro (P<0.01). After one week, immunofluorescence staining showed that endothelial regeneration rate obviously enhanced in shear stress+SBP group (P<0.01). The present study demonstrates that shear stress can promote function of endothelial progenitor cells treated with SBP, which improves the vascular injury repair potentials of EPCs.
ObjectiveTo compare the biological features of early and late endothelial progenitor cells (EPCs) by isolating and culturing early and late EPCs from the human peripheral blood so as to find some unique properties of EPCs and to propose a suitable strategy for EPCs identification. MethodsMononuclear cells were isolated from the human peripheral blood using density gradient centrifugation. Then, the cells were inoculated in human fibronectin-coated culture flasks and cultured in endothelial cell basal medium 2. After 4-7 days and 2-3 weeks culture, early and late EPCs were obtained respectively. The morphology, proliferation potential, surface markers, cytokine secretion, angiogenic ability, and nitric oxide (NO) release were compared between 2 types of EPCs. Meanwhile, the human aortic endothelial cells (HAECs) were used as positive control. ResultsThe morphology of early and late EPCs was different:early EPCs formed a cell cluster with a spindle shape after 4-7 days of culture, and late EPCs showed a cobblestone appearance. Late EPCs were characterized by high proliferation potential and were able to form capillary tubes on Matrigel, but early EPCs did not have this feature. Both types EPCs could ingest acetylated low density lipoprotein and combine with ulex europaeus Ⅰ. Flow cytometry analysis showed that early EPCs did not express CD34 and CD133, but expressed the CD14 and CD45 of the hematopoietic stem cell markers;however, late EPCs expressed CD31 and CD34 of the endothelial cell markers, but did not express CD14, CD45, and CD133. By RT-PCR analysis, the expressions of vascular endothelial growth receptor 2 and vascular endothelial cadherin in early EPCs were significantly lower than those in the late EPCs and HAECs (P<0.05), but no significant difference was found in the expression of von Willebrand factor and endothelial nitric oxide synthase (eNOS) between 2 type EPCs (P>0.05). The concentrations of vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8 were significantly higher in the supernatant of early EPCs than late EPCs (P<0.05). Western blot assay indicated eNOS expressed in both types EPCs, while the expression of eNOS in late EPCs was significantly higher than early EPCs at 5 weeks (P<0.05). Both cell types could produce similar amount of NO (P>0.05). ConclusionThe expression of eNOS and the production of NO could be used as common biological features to identify EPCs, and the strategy of a combination of multiple methods for EPCs identification is more feasible.
ObjectiveTo observe the effects of aquaporin 1 (AQP1) on the proliferation and migration of endothelial progenitor-endothelial progenitor cells (EPC).MethodsBone marrow cells of AQP1 wild-type (WT) (n=6) and knockout-type (KO) mice (n=6) were isolated and differentiated into EPC in vitro. Immunofluorescence was used to detect cell surface antigens to identify EPC. Live cell kinetic imaging and quantification technology, transwell migration assays, as well as scratch test were used to compare the function of EPC between AQP1 WT and KO mice.ResultsEPC culture showed that cells were initially suspended and gradually adhered to typical mesenchymal stem cells within 7 days. After cultured on special medium for endothelial cells they were adhered and differentiated, and fusiform or polygonal, paving stone-like EPC were observed around 14 days. When cultured by special medium of EPC, CD133 and CD31 were positively detected after 7 days, and CD34 and Flk-1 were positively detected after 14 days. Positive expression of AQP1 was only detected in EPC of AQP1 WT mice. Functional studies of EPC revealed there was no significant difference in the proliferation of EPC between AQP1 WT and KO group mice. Transwell assay showed that EPC migration ability of AQP1 KO mice was significantly weaker than that of WT mice. The scratch healing ability of EPC in AQP1 KO mice was significantly lower than that of WT mice.ConclusionsEPC initially shows the characteristics of stem cells and with the prolongation of culture time, EPC gradually shows the characteristics of endothelial cells. AQP1 affects the EPC migration rather than proliferation.
Objective To observe the effects of Galectin-3 on proliferation of vascular endothelial cells derived from peripheral blood endothelial progenitor cells. Methods The cultured peripheral blood endothelial progenitor cells in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cells. Then the cells were cultivated with the galectin-3 of different concentrations, and to observe the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells. Results The abilities of proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells of 0.1, 1.0, 2.5, 5.0, and 10.0 μg/ml groups were higher than that of 0 μg/ml group, there were not statistic significance of the differences between the 0.1，1.0, 2.5, and 0 μg/ml groups （P＞0.05）. But the abilities of proliferation of 5.0 and 10.0 μg/ml groups were obviously higher than that of 0, 0.1, 1.0, and 2.5 μg/ml groups （P＜0.05）, and the abilities of proliferation of 10.0 μg/ml group was also higher than that of 5.0 μg/ml group （P＜0.05）. Conclusion Galectin-3 can promote the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cell.
Objective To observe endothelial progenitor cells (EPCs) participating in the formation of neovascularization in lung adenocarcinoma. Methods EPCs were transfected by recombinant adenovirus carrying LacZ gene in optimal transfection concentration, and then EPCs were injected into animal models of lung adenocarcinoma through the tail vein; afterwards, lung tissues were taken out for pathological examination in the 6th, 7th, 8th week respectively. EPCs were observed to take part in the angiogenesis in the lung adenocarcinoma through X-gal chromogenic dye. Results The optimal multiplicity of infection (MOI) of AD5F35LacZ transfected EPCs was 400. When MOI was 400, maximum transfection efficiency was 97.13±2.08. After 2 weeks, LacZ gene-transfected EPCs began to proliferate in vitro culture, then the EPCs were transplanted into animal models of lung cancer to be involved in the neovascularization formation in the 8th week after transplantation. Conclusion EPCs are involved in the formation of tumor neovascularization after transplantation.