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 investigate the effect of keratin 17 (K-17) on the migration, prol iferation and tube formation of human umbil ical vein endothel ial cell (HUVEC), and to real ize the role of K-17 in angiogenesis. Methods After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and Ncontrol-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell prol iferation abil ity was detected by cell counting. After 30-hour culture, the cell’s abil ities of migration and differentiation to tube were detected by 24-well Mill icell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. Results After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the prol iferation, compared with both control and negative control groups (P gt; 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Mill icell wells within 24 hours (3719.0 ± 319.0) was smaller than both control (7 437.5 ± 212.0) and negative control (7 356.3 ± 795.7) groups, with significant difference (P lt; 0.01). However, there was no significant difference between the control group and the negative control group (P gt; 0.05). After HUVEC were transfected with K-17- siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 ± 0.5), (3.6 ± 0.5) and (3.2 ± 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P lt; 0.01), and there was no significant difference between the negative control group and the control group (P gt; 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 ± 0.02, 0.08 ± 0.01 and 0.72 ± 0.03, respectively. There was significant difference among these three groups (P lt; 0.01). Conclusion K-17 has no impact on cell prol iferation, but may augment endothel ial cell migration, which may facil itate angiogenesis.
【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.
Objective To develop an in vitro three-dimensional angiogenesis system and analyze the expression and function of CD105 in angiogenesis. Methods After primary human umbilical vein endothelial cells (HUVEC) were purified and cultured, the microcarriers were coated with HUVEC and then embedded and cultured into fibrin gel. The angiogenesis process of HUVEC on the microcarriers was formed. The expression of CD105 during this process was detected by reverse transcription polymerase chain reaction (RT-PCR). Antisense oligodeoxynucleotide (ASODN) was used to inhibit the expression of CD105 and the changes of the angiogenesis process were analyzed quantitatively. Results HUVEC on the microcarriers which were embedded into the fibrin gel, occurred the angiogenesis process of sprouts, branches and capillary networks with lumina. During this process, CD105 was over expressed in the periods of forming sprouts and branches, and depressed in the relatively steady periods including the periods before forming sprouts and after forming capillary networks. While the expression of CD105 was inhibited by ASODN, the angiogenesis process was significantly inhibited. Conclusions The expression of CD105 is varied within the angiogenesis process, over expressing during the sprouts and branches forming periods. Inhibiting the expression of CD105 could efficiently inhibit angiogenesis.
ObjectiveTo investigate whether exosomes derived from miR-27a-overexpressing human umbilical vein endothelial cells (HUVECs)—exo (miR-27a) can promote bone regeneration and improve glucocorticoids (GC) induced osteonecrosis of femoral head (ONFH) (GC-ONFH).MethodsThe exo (miR-27a) were intended to be constructed and identified by transmission electron microscopy, nanoparticle tracking analysis, Western blot, and real-time fluorescent quantitative PCR (qRT-PCR). qRT-PCR was used to evaluate the effect of exo (miR-27a) in delivering miR-27a to osteoblasts (MC3T3-E1 cells). Alkaline phosphatase staining, alizarin red staining, and qRT-PCR were used to evaluate its effect on MC3T3-E1 cells osteogenesis. Dual-luciferase reporter (DLRTM) assay was used to verify whether miR-27a targeting Dickkopf WNT signaling pathway inhibitor 2 (DKK2) was a potential mechanism, and the mechanism was further verified by qRT-PCR, Western blot, and alizarin red staining in MC3T3-E1 cells. Finally, the protective effect of exo (miR-27a) on ONFH was verified by the GC-ONFH model in Sprague Dawley (SD) rats.ResultsTransmission electron microscopy, nanoparticle tracking analysis, Western blot, and qRT-PCR detection showed that exo (miR-27a) was successfully constructed. exo (miR-27a) could effectively deliver miR-27a to MC3T3-E1 cells and enhance their osteogenic capacity. The detection of DLRTM showed that miR-27a promoted bone formation by directly targeting DDK2. Micro-CT and HE staining results of animal experiments showed that tail vein injection of exo (miR-27a) improved the osteonecrosis of SD rat GC-ONFH model.Conclusionexo (miR-27a) can promote bone regeneration and protect against GC-ONFH to some extent.
Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0.5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs), we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.
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.
OBJECTIVE: To elongate the proliferation life-span of human umbilicus vein endothelial cell (HUVEC). METHODS: We synthesized the human telomerase reverse transcriptase mRNA (hTERT mRNA) by in vitro transcription, then transferred the hTERT mRNA into HUVEC in quicent stage by lipofect introduction. RESULTS: Telomerase expressed transiently in HUVEC, and the cell life-span was elongated for 7 population doublings. CONCLUSION: Telomerase can be reconstructed controllably and transiently in HUVEC by hTERT mRNA introduction, this method has the potential to be used to elongate the lifespan of cells cultured in vitro.
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.
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.