Objective To investigate the role of calcium- and integrin-binding protein-1（CIB1） in oxidized lowdensity lipoprotein（OX-LDL） inhibiting migration of mouse macrophages. Methods To silence CIB1 express of mouse macrophages by RNA interference, then incubating mouse macrophages with OX-LDL, cell migration and cell spreading of mouse macrophages were analyzed. Results At 24-72h after macrophages transfected CIB1 siRNA, the express of CIB1 protein was restrained obviously. To silence CIB1 express could increase migration and spreading of mouse macrophages significantly. Conclusions CIB1 plays the important role in intracellular modulating mechanism of OX-LDL inhibiting mouse macrophages migration.
ObjectiveTo observe the effects of aquaporin 1 (AQP1) on the proliferation and migration of endothelial progenitor-endothelial progenitor cells (EPC).MethodsBone marrow cells of AQP1 wild-type (WT) (n=6) and knockout-type (KO) mice (n=6) were isolated and differentiated into EPC in vitro. Immunofluorescence was used to detect cell surface antigens to identify EPC. Live cell kinetic imaging and quantification technology, transwell migration assays, as well as scratch test were used to compare the function of EPC between AQP1 WT and KO mice.ResultsEPC culture showed that cells were initially suspended and gradually adhered to typical mesenchymal stem cells within 7 days. After cultured on special medium for endothelial cells they were adhered and differentiated, and fusiform or polygonal, paving stone-like EPC were observed around 14 days. When cultured by special medium of EPC, CD133 and CD31 were positively detected after 7 days, and CD34 and Flk-1 were positively detected after 14 days. Positive expression of AQP1 was only detected in EPC of AQP1 WT mice. Functional studies of EPC revealed there was no significant difference in the proliferation of EPC between AQP1 WT and KO group mice. Transwell assay showed that EPC migration ability of AQP1 KO mice was significantly weaker than that of WT mice. The scratch healing ability of EPC in AQP1 KO mice was significantly lower than that of WT mice.ConclusionsEPC initially shows the characteristics of stem cells and with the prolongation of culture time, EPC gradually shows the characteristics of endothelial cells. AQP1 affects the EPC migration rather than proliferation.
ObjectiveTo investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. MethodsThe survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×103 procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×103) and concomitant increased expression of its shorter proapoptotic forms (20×103). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). ConclusionTSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.
ObjectiveTo observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. MethodsTo construct the adenovirus vector of recombinant Tum5 gene (rAd-Tum5), and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose (HG)-control group (HG group), empty expression vector group (HG+rAd-GFP), and HG+rAd-Tum5 group. Western blot was used to detect the expression of Tum5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. ResultsThe adenovirus vector of recombinant Tum5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation, migration and tubing of the RF/6A cell (F=44.484, 772.666, 137.696;P < 0.05). The comparison of each group indicated that the HG group was higher than normal group (P < 0.05). There were no statistical differences between HG group and HG+rAd-GFP group (P > 0.05). However, the HG+rAd-Tum5 group was less than HG group (P < 0.05), and the same to HG+rAd-GFP (P < 0.05). ConclusionThe adenovirus vector of recombinant Tum5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
ObjectiveTo observe the effects of NDRG1 on proliferation, migration and lumen formation of retinal vascular endothelial cells (RF/6A cells) in monkeys under high glucose condition. MethodsRF/6A cells were divided into normal group, mannitol group, high glucose group, small interfering RNA (siRNA) negative control group without target gene (siRNA group), 30 nmol/L siRNA down-regulated NDRG1 genome (siNDRG1 group) and 50 nmol/L siNDRG1 group. Normal group cells were cultured conventionally. The mannitol group was added with 25 mmol/L mannitol, and the high-glucose group was added with 25 mmol/L glucose. In the siRNA group, 25 mmol/L glucose was added, and then blank siRNA was added for induction. The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1, respectively. All cells were incubated for 24 h for follow-up experiments. Cell proliferation was observed by 4', 6-diaminidine 2-phenylindole staining. Cell counting kit-8 staining was used to detect cell activity. The expression level of NDRG1 mRNA and protein was detected by Western blot and real-time quantitative polymerase chain reaction. Cell migration was observed by cell scratch assay. Cell lumen formation assay was used to detect lumen formation. The two-tailed Student t test was used to compare the two groups. One-way analysis of variance was used to compare groups. ResultsThere were significant differences in cell proliferation rate (t=36.659, 57.645) mobility rate (t=24.745, 33.638) and lumen formation number (t=41.276, 22.867) between high glucose group and normal group and mannitol group (P＜0.01). Compared with normal group and mannitol group, the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased, with statistical significance (t=46.145, 21.541, 36.738, 32.976; P＜0.001). Compared with the siRNA negative group, the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased, and the differences were statistically significant (t=44.275, 40.7577, 57.167, 25.877; P＜0.01). Compared with normal group and siRNA group, cell mobility in 30 nmol/LsiNDRG1 group was increased, and the difference was statistically significant (t=57.562, 49.522; P＜0.01). Compared with normal group and siRNA group, the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision, and the difference was statistically significant (t=63.446, 42.742; P＜0.01). ConclusionDown-regulation of NDRG1 gene can improve the activity, migration and lumen formation of RF/6A cells under hyperglycemia.
Objective Tissue engineered bone implanted with sensory nerve can effectively promote angiogenesis and repair of bone defects. To investigate the effects of calcitonin gene-related peptide (CGRP) on proliferation and migration of human umbilical vein endothelial cells (HUVECs) for further revealing the mechanism of tissue engineered bone implanted with sensory nerve promoting angiogenesis. Methods HUVECs were collected from human umbilical core, and identified through von Willebrand factor (vWF) and CD31 immunofluorescence. The HUVECs were treated with CGRP and were ivided into 6 groups according to CGRP concentration: group A (0 mol/L), group B (1 × 10—12 mol/L), group C (1 × 10—11 mol/L), group D (1 × 10—10 mol/L), group E (1 × 10—9 mol/L), and group F (1 × 10—8 mol/L). The expression of the CGRP1 receptor (CGRP1R) was observed in HUVECs by cell immunofluorescence. The growth rate of HUVECs was detected through AlarmarBlue at 1, 2, 3, 4, and 5 days. Transwell chamber was used to detect the abil ity of cell migration. ELISA assay was used to detect the vascular endothel ial growth factor (VEGF) secretion and the protein expression of focal adhesion kinase (FAK) was examined using Western blot. Results HUVECs were identified through morphology, vWF and CD31 immunofluorescence. HUVECs expressed CGRP1R. CGRP could stimulate HUVECs prol iferation in a time- and concentration-dependent manners; the cell growth rates of groups B-F were significantly higher than that of group A at all time (P lt; 0.05); group F had highest cell growth rate. The number of cell migration of group B-F was significantly higher than that of group A (P lt; 0.05), which increased more than 3 times. Groups B-F had higher amount of VEGF than group A (P lt; 0.05), and groups C and D had highest amount of VEGF. FAK expression of groups B-F was significantly increased at 3, 7, and 10 days after CGRP treatment when compared with group A (P lt; 0.05). Conclusion CGRP may enhance the proliferation and migration of HUVECs by increasing the secretion of VEGF and expression of FAK.
Central venous stenosis is a common complication following long-term dialysis catheter placement in dialysis patients. Generally, percutaneous angioplasty is the treatment of choice, and venous stent implantation should be considered in different situations. However, the venous stent migrating into right atrium is a rare but fatal complication. We presented a patient whose superior vena cava stents migrated into right atrium, resulting in acute tamponade, and exploratory thoracotomy was proceeded.
Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552 (miR-552) on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods ① The MKN-45 cells were divided into MKN-45 blank control group (no transfection), MKN-45 miR-552 inhibitor group [transfection of plasmid inhibiting mir-552 expression (mir-552 inhibitor plasmid)], and MKN-45 negative control group [transfection of negative control plasmid (empty plasmid)], the exosomes were extracted, purified, and identified. Western blotting was used to detect the protein expression of exosomal markers [CD63, CD9, and tumor susceptibility gene 101 (TSG101)]. ② The HUVEC cells were divided into HUVEC control group (added PBS), HUVEC-exosome group (co-cultured with exosomes of MKN-45 cell), HUVEC-negative control exosome group (co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid), and HUVEC-miR-552 inhibitor exosome group (co-cultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid), exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells. Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552, the MTT method was used to detect the proliferation of HUVEC cells, the Transwell chamber method was used to detect the migration of HUVEC cells, the angiogenesis test was used to detect the angiogenesis ability. Results This study successfully extracted exosomes from MKN-45 gastric cancer cells. Observed by transmission electron microscope, the exosomes were all round or elliptical, with a diameter of 100–150 nm, and the exosomal vesicle structure could be seen. Western blotting detection showed that the surface markers of exosomes (CD63, CD9, and TSG101 protein) were expressed in exosomes. The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells. After MKN-45 cells were transfected with miR-552 inhibitor plasmid, compared with the MKN-45 blank control group and MKN-45 negative control group, the relative expression level of miR-552 in the exosomes decreased (P<0.05). Compared with the HUVEC control group, the cell proliferation rate at 24, 48 and 74 h increased, as well as number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-exosomes group increased (P<0.05). Compared with the HUVEC-negative control exosome group, the cell proliferation rate at 24, 48 and 74 h decreased, as well as the number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased (P<0.05). Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation, migration, and angiogenesis of HUVEC cells.
ObjectiveTo investigate the effect of Wnt5a derived from tumor-associated fibroblasts （CAFs） on the migration and invasion of gastric cancer cells. MethodsThe differentially expressed genes Wnt5a between CAFs and normal gastric fibroblasts (NGFs) in gastric cancer tissues and their corresponding normal gastric tissues using the GEO database GSE194261 dataset were screened. Immunohistochemical method was used to detect the expression of Wnt5a protein in tissue samples of clinical gastric cancer patients, and the relationship between Wnt5a protein expression and clinicopathological features of gastric cancer was analyzed. CAFs and NGFs were extracted from fresh surgical specimens of gastric cancer patients, and the expression of Wnt5a in CAFs was detected by real-time fluorescence quantitative-polymerase chain reaction and Western blot experiment. Transwell invasion and migration experiment was used to observe the effects of CAFs, inhibition of Wnt5a expression in CAFs and different concentrations of recombinant Wnt5a protein on the migration and invasion ability of gastric cancer MGC-803 and MKN-28 cell lines in vitro. ResultsThrough the screening of GEO database GSE194261 data set, it was found that Wnt5a was more expressed in CAFs than NGFs (P<0.05). Immunohistochemical results showed that the expression of Wnt5a protein in gastric cancer tissues was significantly stronger than that in normal gastric tissues (P<0.05), and the expression of Wnt5a protein was related to T stage of tumor (χ2=5.035, P<0.05), but not related to gender, age, degree of tumor differentiation, lymph node metastasis, vascular invasion and nerve invasion (P>0.05). Inhibiting Wnt5a derived from CAFs could inhibit the invasion and migration of gastric cancer cells. By stimulating gastric cancer cells with different concentrations of human recombinant Wnt5a protein, it was found that when the concentration of human recombinant Wnt5a protein was greater than 100 ng/mL, the invasion and migration abilities of MGC-803 and MKN-28 gastric cancer cells were significantly increased (P<0.05). ConclusionWnt5a is highly expressed in CAFs derived from the interstitial tissue of gastric cancer, which is related to the invasion depth of gastric cancer and can promote the invasion and migration of gastric cancer cells.