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 assess the safety of intravenous infusion with self-made perfluorocarbon emulsion (PFE) in rats.Methods 35 Wistar rats were randomly divided into a normal control group and six PFE groups (n=5 in each group).The control group was intravenous injected with normal saline and six PFE groups with PFE.Blood samples were obtained from the PFE groups 5 min after injection via vena orbitalis for perfluorocarbon (PFC) measurement.The control group were sacrificed at 2 hours after injection，and the six PFE groups were respectively killed at 2 h，4 h，6 h，24 h，48 h and 10 d after injection.The blood levels of PFC，PaO2，GPT，GOT，BUN and Cr were measured.Pathological changes of lung，liver and kidney were also observed.Results All rats were in good health state after PFE intravenous injection with no obvious pathological changes in liver and kidney.PFC was wide distributed throughout the pulmonary alveoli and capillaries.The levels of GPT and GOT were higher in the PFC groups at 2，4，6 and 24 h than which in the control group (all Plt;0.05)，but there were no significant difference between the PFE 10 d group and the control group.The levels of BUN and Cr had no remarkably differences in all groups.Blood PFC concentrations were (20±1.8)mg/mL，(1.8±0.7)mg/mL，(1.5±0.6)mg/mL，(1.2±0.4)mg/mL，(0.5±0.2)mg/mL，(0.2±0.03)mg/mL，0 mg/mL in the PFE groups respectively at 5 min，2 h，4 h，6 h，24 h，48 h，10 d after injection.PaO2 of the PFE 2 h group was significant higher than which in the control group [(119.2±8.6)mm Hg vs (99.6±4.7)mm Hg，Plt;0.05].Conclusion Intravenous injection with PFE (10 mL/kg) can enhance PaO2 with good safety and no obvious influence on pathology of lung，liver and kidney，so may be used for the treatment study of acute lung injury.
Abstract: Intravenous leiomyomatosis (IVL) is a rare kind of uterine myoma. It is a benign smooth muscle tumor with invading growth pattern. The tumor extends into venous channels, but rarely invades tissues. It grows along the refluxing direction of the venous channels, uterine vein, ovarian vein, and beyond the uterus, extends into the inferior vena cava till the right atrium or pulmonary arteries, resulting in intracardiac leiomyomatosis (ICL). At present, the tumor can be detected by ultrasonic waves, computer tomography and magnetic resonance imaging. The main ICL therapy is surgery which is divided into onestage operation and twostage operation in which the key is the complete tumor excision. Most sufferers have a good prognosis, but there are possibilities of recurrence. Missed diagnosis and misdiagnosis are not uncommon, because the disease is rare with hided and diversified clinical manifestations. It is fatal without special characteristics. For a better understanding of ICL, the recent research and treatment of ICL are reviewed.
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 construct human recombinant lentiviral expression vector of microRNA-210 (miR-210)and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12 (HUVE-12). Methods The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein (GFP)-pre-miR-210 wasconstructed by molecular cloning and transfected to HUVE-12 (LV-miR-210-GFP group), only pGCSIL-GFP was transfectedas control group (LV-GFP group). The miR-210 expression activity was evaluated by GFP reporter through fluorescencedetection and real-time fluorescent quantitative PCR. The ephrinA3 protein expression was measured by flow cytometry. Theconcentration of vascular endothelial growth factor (VEGF) in culture supernatant was determined by ELISA. The cells werecultured in 96-well culture plate coated with Matrigel to assess the abil ity of capillary formation. Results The recombinantplasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing. Fluorescencedetection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection. Real-timefluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than thatin LV-GFP group (t= —11.10，P=0.00). Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-210-GFP group (12.52% ± 0.67%) was significantly lower than that in LV-GFP group (73.22% ± 1.45%) (t= —66.12，P=0.00).The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group[(305.29 ± 16.52) pg/mL vs. (42.52 ± 3.11) pg/mL, t= —27.06，P=0.00]. In vitro capillary-l ike formation assay showed that thenumber of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group (17.33 ± 6.33 vs. 6.33 ± 2.33,t= —2.83，P=0.04). Conclusion The recombinant lentiviral expression vector of miR-210 is constructed successfully andHUVE-12 over-expressing miR-210 can significantly increase the capillary formation, which facil itates further study on themolecular functions of miR-210 in angiogenesis.
Objective To investigate the biological response and chemotaxis of endothel ial cells on template materials with different protein concentrations on the same surface, to provide the evidence for deep understanding of chemical induced cell motil ity. Methods Microcontact printing technique was employed to fabricate template materials with four different concentrations of collagen (50, 100, 200, 300 μg/mL) on the same substrate. Scanning electron microscopy was employed to characterize the qual ity of polydimethylsiloxane (PDMS) stamp. Confocal laser scanning microscopy (CLSM) was util ized to characterize the absorption of different concentrations of FITC conjugated collagen (50, 100, 200, 300 μg/mL) on the substrates surfaces. Software was used to analyze the fluorescence intensity of adsorbed protein on the substrates. Albumin was then used to block the substrates for cell culture of human umbil ical vein endothel ial cells (hUVEC). Substrates with no collagen adsorption were used as control samples. The influence of different concentrations of collagen on the prol iferation of hUVEC was investigated via MTT assay at 6, 24, 48 and 72 hours of culture. The cytoskeletal structures of cells were characterized by CLSM. The cell’ s migration speed and absolute displacement were measured by path measurement of single cell after 24 hours of culture. Results Fabricated PDMS stamps with complete pattern were flat. Template substrates were fully covered with evenly distributed collagen protein. The fluorescence intensities were 38.51 ± 1.63, 55.21 ± 3.88, 73.17 ± 3.59, and 80.95 ± 1.12 in adsorbed FTIC conjugated collagen with 50, 100, 200 and 300 μg/mL, respectively. Endothel ial cells spread better on various substrates coated with collagen than those of control samples. The prol iferation of endothel ial cells on collagen coated substrateswas significantly higher than that of control group (P lt; 0.05). With collagen concentration increasing from 50 µg/mL to 300µg/mL, the prol iferation abil ities and absolute displacements of endothel ial cells significantly increased (P lt; 0.05). Except for the group with 300 μg/mL, the migration speed of endothel ial cells on collagen coated substrates was significantly lower (P lt; 0.05) than that of control group. However, the migration speed of endothel ial cells on collagen coated substrates significantly increased (P lt; 0.05) along with collagen concentration increasing from 50 µg/mL to 300 µg/mL. Conclusion It is feasible to acquire domains with different protein concentrations on the same substrate using microcontact printing technique for investigating cell’s chemotaxis.
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.
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.