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.
Objective To compare canine decel luarized venous valve stent combining endothel ial progenitor cells (EPC) with native venous valve in terms of venous valve closure mechanism in normal physiological conditions. Methods Thirty-six male hybrid dogs weighing 15-18 kg were used. The left femoral vein with valve from 12 dogs was harvested to prepare decelluarized valved venous stent combined with EPC. The rest 24 dogs were randomly divided into the experimental group and the control group (n=12 per group). In the experimental group, EPC obtained from the bone marrowthrough in vitro ampl ification were cultured, the cells at passage 3 (5 × 106 cells/mL) were seeded on the stent, and the general and HE staining observations were performed before and after the seeding of the cells. In the experimental group, allogenic decelluarized valved venous stent combined with EPC was transplanted to the left femoral vein region, while in the control group, the autogenous vein venous valve was implanted in situ. Color Doppler Ultrasound exam was performed 4 weeks after transplantation to compare the direction and velocity of blood flow in the distal and proximal end of the valve, and the changes of vein diameter in the valve sinus before and after the closure of venous valve when the dogs changed from supine position to reverse trendelenburg position. Results General and HE staining observations before and after cell seeding: the decelluarized valved venous stent maintained its fiber and collagen structure, and the EPC were planted on the decelluarized stent successfully through bioreactor. During the period from the reverse trendelenburg position to the starting point for the closure of the valve, the reverse flow of blood occurred in the experimental group with the velocity of (1.4 ± 0.3) cm/s; while in the control group, there was no reverse flow of blood, but the peak flow rate was decreased from (21.3 ± 2.1) cm/s to (18.2 ± 3.3) cm/s. In the control group, the active period of valve, the starting point for the closure of the valve, and the time between the beginning of closure and the complete closure was (918 ± 46), (712 ± 48), and (154 ± 29) ms, respectively; while in the experimental group, it was (989 ± 53), (785 ± 43), and (223 ± 29) ms, respectively. There was significant difference between two groups (P lt; 0.05).After the complete closure of valve, no reverse flow of blood occurred in two groups. The vein diameter in the valve sinus of the experimental and the control group after the valve closure was increased by 116.8% ± 2.0% and 118.5% ± 2.2%, respectively, when compared with the value before valve closure (P gt; 0.05). Conclusion Canine decelluarized venous valve stent combined with EPC is remarkably different from natural venous valve in terms of the valve closure mechanism in physiological condition. The former rel ies on the reverse flow of blood and the latter is related to the decreased velocity of blood flow and the increased pressure of vein in the venous sinus segment.
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 establish the three diamension-model and to observe the contribution of endothelial progenitor cell (EPC) in the angiogenesis and its biological features. MethodsEPC was obtained from the rats’ peripheral blood. Its cultivation and amplification in vitro were observed, and the function of the cultural EPC in vitro was detected. The three diamension-model was established and analyzed. ResultsEPC was obtained from the peripheral blood successfully. The proliferation of the EPC which induced with VEGF(experimental group) was better than that without VEGF （control group） at every different phase (P＜0.01). It was found that EPC grew into collagen-material from up and down in the three diamension-model, and its pullulation and infiltration into the collagen were seen on day 1 after cultivation. With the time flying, there were branch-like constructions which were vertical to the undersurface of collagen and interlaced to net each other. It showed that in experimental group the EPC grew fast, its infiltration and pullulation also were fast, the branch-like construction was thick. But in control group, the EPC grew slowly, infiltration and pullulation were slow, the branch-like construction was tiny and the depth of infiltration into collagen was superficial. The number of new vessels in experimental group was larger than that in the control group at every different phase (P＜0.01). ConclusionRat tail collagen can induce EPC involved in immigration, proliferation and pullulation in angiogenesis. The three-diamension model of EPC can be used to angiogenesis research. VEGF can mobilize and induce EPC to promote the angiogenesis.
Objective To measure the level of circulating endothelial progenitor cells ( EPCs) in peripheral blood of patients with acute exacerbation of chronic obstructive pulmonary disease ( AECOPD) , and to explore the relationship between EPCs and severity markers of the disease and cardiovascular adverse outcome predictors.Methods Forty patients with COPD were recruited, including 27 at acute exacerbation phase and 13 with stable COPD from December 2010 to December 2011. Sixteen healthy nonsmokers were included as controls. Circulating EPCs were isolated by Ficoll density-gradient centrifugation and purified by Magnetic Activated Cell Sorting system. High-sensitivity C-reactive protein ( hsCRP) was estimated by using a latex immunoturbidimetric assay kit, and matrix metalloproteinase-9 ( MMP-9) was measured by enzymelinked immunosorbent assay ( ELISA) . Arterial blood gas analysis and echocardiograph were performed in the AECOPD patients. The correlations between circulating EPCs, lung function, and cardiovascular markers were investigated. Results Circulating EPCs were significantly lower in AECOPD and stable COPD patients compared with the healthy controls [ ( 5.1 ±2.6) ×103 /mL and ( 6.0 ±3.2) ×103 /mL vs. ( 9.0 ±4.3) × 103 /mL, Plt;0. 05] . EPCs had a weak correlation with hsCRP ( P = 0. 033) , but not with MMP-9. In the AECOPD patients, EPC counts were significantly inversely correlated with PASP ( pulmonary artery systolic pressure) and NT-proBNP ( amino-terminal pro-brain natriuretic peptide) levels, and positively with left ventricular ejection fraction. No correlations were found between EPCs and lung function, blood gas, hospital stays or smoking index. Conclusions Circulating EPCs were significantly lower in AECOPD patients compared with healthy controls, in which systemic inflammation might be involved. Decreased EPCs were correlated with cardiac dysfunction in patients with AECOPD, which may account for the increased cardiovascular risk in this population.
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 research the magnetic labeled endothelial progenitor cells(EPCs) transplanted into the rat of venous thrombosis model through the tail vein and track transplanted stem cells in vivo, provide an effective monitoring technology for promoting organization and recanalization of deep venous thrombosis. MethodsBone marrowderived EPCs were extracted, purified, and identified, then labeled with the new SPIO particles. At the same time, the inferior vena cava thrombus models in rats were made, which were randomly divided into four groups:SPIO group (EPCs labeled with SPIO transplantation), Dil group (EPCs labeled with Dil transplantation), control group (simple EPCs transplantation), and blank control group (1 mL medium transplantation). After transplantation, the MRI, HE staining, and immunohistochemical staining were performed and the capillary density was counted under high-power microscope. ResultsThe MRI showed that EPCs labeled with SPIO had migrated to the inferior vena cava thrombus and the mass of high signal shade was seen, with the extension of time, the signal strengthened gradually. On day 14-21, the signal became the strongest, then decreased gradually. The immunohistochemical staining and HE staining showed that there were a mass of the new capillary in the specimens of thrombus of the SPIO group, Dil group, and control group, the difference was not statistically significant among these three groups(P > 0.05), but which was significant difference as compared with blank control group (P < 0.05). ConclusionCompared with EPCs labeled with Dil, it
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.
ObjectiveTo review the research progress of the co-culture system for constructing vascularized tissue engineered bone. MethodsThe recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. ResultsThe construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. ConclusionThe strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.