ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation. MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro. ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group. ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
Objective To investigate the protocols of combined culture of human placenta-derived mesenchymal stem cells (HPMSCs) and human umbilical vein endothelial cells (HUVECs) from the same and different individuals on collagen material, to provide the. Methods Under voluntary contributions, HPMSCs were isolated and purified from human full-term placenta using collagenase IV digestion and lymphocyte separation medium, and confirmed by morphology methods and flow cytometry, and then passage 2 cells were cultured under condition of osteogenic induction. HUVECs were isolated from fresh human umbilical vein by collagenase I digestion and subcultured to purification, and cells were confirmed by immunocytochemical staining of von Willebrand factor (vWF). There were 2 groups for experiment. Passage 3 osteoblastic induced HPMSCs were co-cultured with HUVECs (1 ∶ 1) from different individuals in group A and with HUVECs from the same individual in group B on collagen hydrogel. Confocal laser scanning microscope was used to observe the cellular behavior of the cell-collagen composites at 1, 3, 5, and 7 days after culturing. Results Flow cytometry showed that HPMSCs were bly positive for CD90 and CD29, but negative for CD31, CD45, and CD34. After induction, alizarin red, alkaline phosphatase, and collagenase I staining were positive. HUVECs displayed cobble-stone morphology and stained positively for endothelial cell marker vWF. The immunofluorescent staining of CD31 showed that HUVECs in the cell-collagen composite of group B had richer layers, adhered and extended faster and better in three-dimension space than that of group A. At 7 days, the class-like microvessel lengths and the network point numbers were (6.68 ± 0.35) mm/mm2 and (17.10 ± 1.10)/mm2 in group A, and were (8.11 ± 0.62) mm/mm2 and (21.30 ± 1.41)/mm2 in group B, showing significant differences between the 2 groups (t=0.894, P=0.000; t=0.732, P=0.000). Conclusion Composite implant HPMSCs and HUVECs from the same individual on collagen hydrogel is better than HPMSCs and HUVECs from different individuals in integrity and continuity of the network and angiogenesis.
OBJECTIVE: To explore the possibility of improving the performance of tissue engineering valve by means of preendothelialization with cultured human umbilical vein endothelial cell(hUVEC) and to develop a new xenogenic bioprosthesis valve material. METHODS: The porcine aortic valves treated by use of glutaraldehyde(GA), epoxychloropropane(EC), L-glutamic acid(L-GA) and cellular extraction(CE) respectively were divided into four groups; group 1(GA), group 2(EC), group 3(EC + L-GA), and group 4(EC + L-GA + CE). The cultured hUVECs were seeded onto the treated porcine aortic valve, then that stuff were examined by means of EC VIII factor staining, living cells counting and microscopy. RESULTS: The cultured hUVEC could adhere to culturing bottle wall an hour later, and propagated to two passages after seven days. The cells increased with serial passage at a 7-day interval. But the hUVEC grew slowly when seeded onto the treated valve material except group 4. The cells in group 4 covered the surface of valve completely seven days later, which could also be seen in group 3 but not completely. There was no cell growing in group 1, and only fewer in group 2. The living cell in groups 3 and 4 were significantly more than in groups 1 and 2 on the 3rd, 7th and 14th days (P lt; 0.01), meanwhile, the number of cells in group 4 were also significantly more than that in group 3 (P lt; 0.05). The covering area of cultured cell on the valve material in groups 3 and 4 was significantly larger than that in groups 1 and 2. The covering area of cell in group 4 was over 95%, and higher than that in group 3(60%-70%). The hUVEC of group 4 arranged in pattern of three dimension. So it could resist rising of foreign power from the cardiac cavity of high pressure and flowing volume. There was no cell on the leaflet surface in group 1, and only a few pinch of cells could be seen in group 2. CONCLUSION: The porcine aortic valve can be used to be an ideal xenogeneic valve scaffold; the scaffold of porcine aortic valve should be treated by use of epoxy-chloropropane, L-glutamic acid and cellular extraction, so that a best growing environment to the hUVEC would be given; the cultured hUVECs used to be source of seed living cell had a boundless prospects; the growing velocity of cultured hUVEC was controllable, which facilitated clinical application; and the endothelial cells of xenogeneic valve material which grew compactly onto the scaffold can resist rising of foreign power from the cardiac cavity itself.
【摘要】 目的 通过比较两种原代人脐静脉内皮细胞的分离培养方法并对细胞特异性抗原进行鉴定,探索提高原代内皮细胞体外培养存活率及纯化率的方法。 方法 采用一次性无菌注射器向人脐静脉灌注消化液,消化液的浓度和消化时间分别025%(质量体积比)胰蛋白酶,10 min和01%(质量体积比)胶原酶Ⅱ,15 min。通过在倒置显微镜下观察细胞的形态特点和用免疫荧光染色的方法对细胞进行鉴定,比较两种消化方法的优劣。 结果 01%胶原酶Ⅱ,15 min的消化方法较025%胰蛋白酶,10 min对原代人脐静脉内皮细胞有更好的分离效果,活细胞数量多且细胞纯度较高。免疫荧光染色结果表明细胞内有Ⅷ因子相关抗原表达。结论 胶原酶Ⅱ可以有效分离脐静脉内皮细胞,最佳消化条件是01%胶原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.
ObjectiveTo construct a lentiviral vector-mediated gene-targeted small interfering RNA (siRNA) vector to vascular endothelial growth factor (VEGF), and choose the RNAi with the highest silence efficiency to VEGFA gene. MethodsThree kinds of VEGFA gene-targeted hairpin siRNA was designed (KD1, KD2, KD3), then two complementary oligo nucleotide strand were synthesized and inserted into pGCSIL-GFP vector. After annealing, the recombined vector pGCSIL-GFP-siVEGFA was gotten, which was digested by restrictive enzyme and sequenced, and was co-transfected with the pHelper 1.0 and pHelper 2.0 into 293T cells by Lipofectamine 2000. After that, the new vector was transfected into human umbilical vein endothelial cells (HUVECs), and the mRNA expression level of VEGFA gene in cells was detected by RT-PCR. Then we compared the mRNA expression level of VEGFA gene of the 3 groups. ResultspGCSIL-GFP-siVEGFA was built successfully, and all the siRNA could silence the expression of VEGFA mRNA in the HUVECs, and the relative expressions of VEGFA mRNA to the control group were 0.614±0.043 (KD1), 0.334±0.030 (KD2), and 0.201±0.015 (KD3) respectively. ConclusionWe've successfully constructed the siRNA vector for VEGFA mRNA, which can obviously suppress the expression of VEGFA mRNA.
ObjectiveTo compare the different effects of ubiquitin(UB) on human umbilical vein endothelial cells (HUVECs) and macrophages under normal circumstances,and analyze whether UB could protect HUVECs from lipopolysaccharide(LPS) induced injury. MethodsThe morphologic changes of HUVECs in vitro with up-rising concentrations of UB interventions were observed. HUVECs and human macrophages in vitro were divided into 4 groups according to UB concentration (0.01 μg/mL,0.1 μg/mL, 1 μg/mL, and 10 μg/mL). Supernatant and cells of each group were collected in 24 h after UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA while NF-κB protein level in cells was detected by Western blot. HUVECs were divided into a LPS group(LPS 10 μg/mL) and an UB+LPS group(UB 0.1 μg/mL,LPS 10 μg/mL). The supernatant of the two groups were collected in 8,16 and 24 h after LPS and UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA. ResultsThe injury of HUVECs got worse with the ascending concentrations of UB.At the concentration of 50 μg/mL,UB induced HUVECs got ballooned and died massively. With the increase of UB concentration,the levels of TNF-α and VCAM-1 in HUVECs' supernatant ascended firstly and then descended,while those in human macrophages' supernatant ascended gradually. zHowever,the tendency of the NF-κB protein level in the two kinds of cells was similar when the concentration of UB increased.At the consentration of 0.1 μg/mL or 1 μg/mL,ubiquitin induced NF-κB protein level obviously increased.At the concentration of 0.01 μg/mL or 10 μg/mL,UB induced the protein level was similar with those of the control group and even decreased slightly. There was no significant difference in TNF-α or VCAM-1 levels at each time point between the LPS group and the UB+LPS group. ConclusionsUB injuries HUVECs obviously at a low concentration but injuires human macrophages at much higher concentraton. UB can not protect HUVECs from LPS-induced injury in vitro.
ObjectiveTo prepare polyurethane (PU) microspheres and evaluate its physicochemical properties and biocompatibility for biomedical applications in vitro. MethodsThe PU microspheres were prepared by self-emulsification procedure at the emulsification rates of 1 000, 2 000, 3 000, and 4 000 r/min. The molecular structure was tested by Fourier transform infrared spectrometer and the surface and interior morphology of PU microspheres were observed by scanning electron microscopy (SEM). PU microspheres prepared at best emulsification rate were selected for the subsequent experiment. The human umbilical vein endothelial cells (HUVECs) were cultured and seeded on the materials, then cell morphology and adhesion status were observed by calcein-acetoxymethylester/pyridine iodide (Calcein-AM/PI) staining. The cells were cultured in the H-DMEM containing 10%FBS with additional 1% phenol (group A), in the extracts of PU prepared according to GB/T 16886.12 standard (group B), and in H-DMEM containing 10%FBS (group C), respectively. Cell counting kit 8 (CCK-8) assay was used to detect the cell viability. The blood compatibility experiments were used to evaluate the blood compatibility, the PU extracts as experimental group, stroke-physiological saline solution as negative control group, and distilled water as positive control group. The hemolytic rate was calculated. ResultsThe SEM results of PU microspheres at the emulsification rate of 2 000 r/min showed better morphology and size. The microstructure of the PU was rough on the surface and porous inside. The Calcein-AM/PI staining showed that the HUVECs attached to the PU tightly and nearly all cells were stained by green. CCK-8 assays demonstrated that group B and group C presented a significantly higher cell proliferative activity than group A (P<0.05), indicating low cytotoxicity of the PU. The absorbance value was 0.864±0.002 in positive control group and was 0.015±0.001 in negative control group. The hemolysis rate of the PU extracts was 0.39%±0.07% (<5%), indicating no hemolysis. ConclusionThe PU microspheres are successfully prepared by self-emulsification. The scaffold can obviously promote cell attachments and proliferation and shows low cytotoxicity and favorable blood compatibility, so it might be an ideal filler for soft tissue.
ObjectiveTo observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR).MethodsTwenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects.ResultsThe expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant (tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant (F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant (F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant (F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant (P＜0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group (P=0.03) and no significant difference in FSTL1 protein expression (P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant (F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups.ConclusionThe expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people.