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BACKGROUND:The quantity and quality of seed cel s is a critical bottleneck of the development of vascular tissue engineering. To address this issue, stem cel-derived endothelial cel s have been a hot spot in this field due to their potential in providing the ideal seed cel s. OBJECTIVE:To elucidate the effect of vascular endothelial growth factor (VEGF) supplementation combined with hypoxic culture condition on the lineage-specific differentiation of embryonic stem cel s into endothelial cel s. METHODS:Serum-free medium mTeSR?1 was applied to cultivate H9 cel s in vitro. A conditioned medium containing 50μg/L vascular endothelial growth factor was utilized to induce H9 cel s to differentiate into endothelial cel s under the hypoxic culture condition (5%O2). The cel under normal condition (5%CO2) with or without vascular endothelial growth factor served as controls. The phenotype and function of human embryonic stem cel s-derived endothelial cel s were assayed by immunofluorescence staining, quantitative RT-PCR, and low-density lipoprotein uptake experiment. RESULTS AND CONCLUSION:Compared with the control group, the H9 cel s were induced to be differentiated into endothelial-like cel s more efficiently when they were cultivated under a conditioned medium with vascular endothelial growth factor supplementation under the hypoxic condition. These differentiated cel s not only expressed some important surface markers of endothelia cel s, including kdr, pecam, but also took in low-density lipoprotein to form microvessle-like structures. This culture system supports a synergy effect of vascular endothelial growth factor and hypoxic environment that can efficiently promotes the lineage-specific differentiation of embryonic stem cel s into endothelial cel s with good phenotype and functionality.
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BACKGROUND:It has been proved that erythropoietin can promotes angiogenesis in injured tissue, which is closely related to the proliferation and differentiation of endothelial progenitor cel s. However, the involved mechanism remains unclear yet. OBJECTIVE:To investigate the effect of erythropoietin on the function and activity of bone marrow-derived endothelial progenitor cel s in mice, and to explore the signal pathway. METHODS:The endothelial progenitor cel s from the bone marrow of mice were separated by means of density gradient centrifugation and then cultured. The cel s were preconditioned by specific inhibitor of PI3K (LY294002), and were divided into the fol owing groups:EGM-2 group, three erythropoietin preconditioned groups (the concentrations of erythropoietin in medium were 1, 5, 10 U/mL respectively), erythropoietin+LY group (10 U/mL erythropoietin and 10 mmol/L LY294002 in medium), LY group (10 mmol/L LY294002 in medium), dimethyl sulfoxide group (1 mL/L dimethyl sulfoxide in medium). The cel proliferation and apoptosis were evaluated by cel counting kit-8 and flow cytometry respectively. The contents of endothelial nitric oxide synthase and vascular endothelial growth factor in cel lysates were detected by the method of ELISA, and the expressions of Akt and p-Akt were by western blot assay. RESULTS AND CONCLUSION:Erythropoietin could promote the proliferation of endothelial progenitor cel s in a dose-dependent manner, which was, however, completely inhibited by LY294002. The apoptosis rate in the erythropoietin preconditioned groups was significantly lower than that in the erythropoietin+LY group. The contents of endothelial nitric oxide synthase and vascular endothelial growth factor in cel lysates of LY group and erythropoietin+LY group were significantly lower than those in the erythropoietin groups. There was no difference in Akt expression found in each group, while the p-Akt expression in the erythropoietin+LY group was significantly lower than that in the erythropoietin groups. The above results reveal that erythropoietin can promote the proliferation of endothelial progenitor cel s and decrease the cel apoptosis, which is depending on PI3K/Akt signal pathway.
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BACKGROUND:With the development of biochemistry and cel biology, fracture has being study deeper, blood supply has been known to be an important factor influencing the fracture healing. Endothelial progenitor cel s with good ability of angiogenesis wil have a good clinical prospect in fracture healing. OBJECTIVE:To review the recent research of endothelial progenitor cel s in fracture healing. METHODS:A computer-based online search of CNKI, Wanfang, PubMed databases was performed to col ect articles published between 1980 and 2014 with the key words“endothelial progenitor cel , fracture, neovascularization, angiogenesis”in Chinese and English. A total of 48 articles addressing endothelial progenitor cel for angiogenesis in fracture healing were included in result analysis. RESULTS AND CONCLUSION:Increasing evidence has shown that endothelial progenitor cel s have great ability of neovascularzition and angiogenesis. Endothelial progenitor cel s used in tissue engineering scaffolds can promote the survival rate of scaffolds in vivo, which is appropriate to a great part of delayed union and nonunion patients. However, the large-scale treatment with endothelial progenitor cel s stil has many problems, such as isolation, culture and amplification of endothelial progenitor cel s in vitro, the number of transplanted cel s and selection of scaffolds for transplanted cel s, which need further research.
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BACKGROUND:Coculture of bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s can improve both osteogenic and angiogenic outcomes and provide a promising strategy for bone tissue engineering and osteanagenesis. OBJECTIVE:To summarize recent researches and related progresses in coculture of human umbilical vein endothelial cel s and bone marrow mesenchymal stem cel s. METHODS:A computer-based online search of CNKI database from January 2000 to March 2012, PubMed database and Web of Knowledge database from January 1980 to March 2012, was performed with the keywords of“human umbilical vein endothelial cel s, bone mesenchymal stem cel s, coculture, tissue engineering”both in Chinese and English. A total of 135 articles were screened out, 103 of them were excluded due to unrelated study objective and repeated contents, and final y 32 articles were involved in further analysis. RESULTS AND CONCLUSION:At present, studies on coculture of bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s mainly focus on mimicking in vivo environments, the interactions between cel s, and the influence of different cel ratios and culture media. Most of these researches play important roles in bone tissue engineering and bone regeneration therapy, but the mechanism of action and concrete regulation in vivo between bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s stil need further research and analysis.
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BACKGROUND:Animal studies have indicated ultrasound-mediated microbubbles can significantly enhance the effect of stem cel transplantation to treat ischemic diseases. But its mechanism is stil unknown. OBJECTIVE:To explore the mechanism of ultrasound-mediated microbubbles to significantly enhance the effect of stem cel transplantation in the treatment of ischemic diseases. METHODS:Bone marrow mesenchymal stem cel s and vascular endothelial cel s of rats were cultured in vitro, and then randomized to three groups:control group with no intervention, ultrasound group exposed to ultrasound at 1 MHz, 1 W/cm2 for 90 seconds, and ultrasound-mediated microbubble group treated with 5μL liposomes ultrasound microbubbles containing fluorocarbon gases (about 2×1011/L) and ultrasound exposure at 1 MHz, 1 W/cm2 for 90 seconds. RESULTS AND CONCLUSION:Compared to the control group, ultrasound-mediated microbubbles significantly increased expressions of vascular endothelial growth factor and stromal cel-derived factor 1 in the supernatant of vascular endothelial cel s (P0.05). These findings suggest that 1 W/cm2 ultrasound-mediated microbubbles can promote vascular endothelial growth factor and stromal cel-derived factor 1 secretion by vascular endothelia cel s, and meanwhile promote CXCR4 gene expression in bone marrow mesenchymal stem cel s. This may be the mechanism of the ultrasound-mediated microbubbles enhancing homing effect of transplanted stem cel s.
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BACKGROUND:Establishment of in vitro culture system of human placental microvascular endothelial cel s with high purity is very important. In recent studies, some scholars have successful y obtained a large number of placental microvascular endothelial cel s by three-stepenzyme digestion and magnetic separation method, but the procedures were extremely complex and it had great damage to the cel s. Therefore, how to separate human placental microvascular endothelial cel s easily and obtain high-purified cel s has become a research hotspot. OBJECTIVE:To investigate an efficient method to isolate and purify human placental microvascular endothelial cel s from early vil us microvessels, observe the cel growth and identify the cel s. METHODS:The vil i from normal early pregnancies (6-8 weeks) after artificial abortion were col ected aseptical y. Using two-step digestion procedure and discontinuous Percol density gradient centrifugation method, human placental microvascular endothelial cel s were obtained. Then the cel s were identified by trypsin digestion method and repeated adherence method. RESULTS AND CONCLUSION:Human placental microvascular endothelial cel s were isolated successful y from early vil i. The primary cel s adhered to the wal s after inoculated for 24 hours and entered logarithmic phase at 10 days. 80%of the cel s achieved a confluence at 12-13 days after inoculating. The subculture cel s grew swiftly with the typical cobblestone appearance. Immunofluorescence staining showed that, cultured human placental microvascular endothelial cel s demonstrated a strong positive reaction to von Wil ebrand factor antigen and CD31, accounting for 100%. MTT assay results showed that, human placental microvascular endothelial cel s at passage 5 exhibited an S-shaped growth curve. High-purity human placental microvascular endothelial cel s can be obtained by proteolytic enzymes digestion and discontinuous Percol density gradient centrifugation method, and the purity is detected by trypsin digestion method and repeated adherence method.