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.
Objective To explore the effects of calcitonin gene-related peptide (CGRP) on the migration of bone marrow mesenchymal stem cells (BMSCs) and vascular endothel ial growth factor (VEGF) expression in vitro. Methods TheBMSCs were isolated from Sprague Dawley rats using whole bone marrow adherence method. At 1, 2, and 3 weeks after culture, the expressions of CGRP receptor (CGRPR) was detected by Western blot. The BMSCs were treated with CGRP at concentration 1 × 10-8 mol/L (experimental group) and did not treated (control group), and the efficacy of BMSCs migration was analyzed by Transwell chamber assay after 72 hours; at 1, 3, 5, and 7 days, the mRNA expressions of vascular cell adhesion molecule 1 (VCAM-1) were detected by real-time fluorescent quantitative PCR; the protein expressions of VEGF were examined using immunohistochemistry and Western blot. Results CGRPR expressed stably in the cultured BMSCs and reached the peak at 2 weeks. CGRP had a significantly enhanced role in promoting cell migration. The number of cell migration was (3.20 ± 1.77) cells/HP in experimental group and (1.11 ± 0.49) cells/HP in control group, showing significant difference (t=4.230, P=0.001). In experimental group, the expressions of VCAM-1 mRNA increased with time and reached the peak at 7 days. There were significant differences in the expressions of VCAM-1 mRNA between control group and experimental group at 3, 5, and 7 days (P lt; 0.05). Immunocytochemistry results showed positive DAB staining for VEGF at 5 and 7 days in experimental group. Western blot results showed that the protein expressions of VEGF increased significantly at 5 and 7 days in experimental group when compared with control group (P lt; 0.05), which was signfiantly higher at 5 days than at 7 days in experimental group (P lt; 0.05). Conclusion CGRP can promote the migration of BMSCs and stimulate the protein expression of VEGF, which may plays an important role in regulating bone metabol ism by increasing angiogenesis.
Objective To investigate the effect of monocyte chemoattractant protein 1 (MCP-1) on the migration of the induced and differentiated mouse bone marrow mesenchymal stem cells (BMSCs) for raising the efficacy of intravenous transplantation of BMSCs. Methods The BMSCs were cultured with the method of differential adhesion and density gradient centrifugation of C57/BL10 mice, and were identified by alkal ine phosphatase Gomori modified staining after osteogenic inducing. At the 3rd passage, the BMSCs were induced to the myoblasts with 5-azacytidine (5-Aza). The chemotaxis of MCP-1 in the induced and differentiated BMSCs in vitro at concentrations of 25, 50, 100, 200, and 400 ng/mL was observed through the migration test, by counting the number of the migrated cells. The expression of the chemokine receptor 2 (CKR-2) in the induced and differentiated BMSCs was detected with the flow cytometry. Results The cells could be cultured with the methods of differential adhesion and density gradient centrifugation and still had higher prol iferative and differentiative potency; the induced cells at the 3rd passage could differenciate to the osteoblasts, confirming that the cells were BMSCs; the myogenic induced BMSCs possesed the sarcotubule structure. The number of the migrating BMSCs at MCP-1 concentrations of 25-400 ng/ mL were respectively 35.066 7 ± 6.584 2, 43.200 0 ± 6.460 8, 44.466 7 ± 4.823 5, 45.600 0 ± 8.650 3, and 50.733 3 ± 7.582 5; showing significant difference when compared with control group (28.333 3 ± 8.917 6, P lt; 0.05), and presenting significant difference among 25, 50, 400 ng/mL groups compared with each other (P lt; 0.05). The expression of CKR-2 in the mouse BMSCs (48.0%) was significantly higher (P lt; 0.001) than those of blank control (0.6%) and negative control (17.0%). Conclusion The results indicate that the MCP-1 can induce the migration of mouse BMSCs by MCP-1/CKR-2 pathway.
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.
Objective To investigate the effects of NGF on the prol iferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound heal ing. Methods The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell prol iferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyprol ine and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. Results Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/ mL) showed that NGF did not influence the prol iferation of HDFs (P gt; 0.05). When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P gt; 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyprol ine and FQ-PCR (P gt; 0.05). The rates of HDFs’ migration at various concentrations of NGF (0, 50, 100, 200 ng/ mL) were 52.12% ± 6.50%, 80.67% ± 8.51%, 66.33% ± 3.58%, and 61.19% ± 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P lt; 0.05). Conclusion NGF does not influence prol iferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts.
Objective To study the function and mechanism of G protein coupled receptor kinase interacting protein 1(GIT1) RNA hairpin (GIT1-RNAh) in osteoblast migration. Methods The sixth passage osteoblasts were divided into 2 groups and were infected by GIT1-RNAh (experimental group) and green fluoresence protein RNA hairpin (GFP-RNAh) (control group) adenovirus for 12 hours respectively. Each group was further classfied into two groups according to with or without platelet-drived growth factor (PDGF) stimulation. The GIT1 expression and Paxillindistribution was analyzed by immunofluorescence staining. Paxillin phosphorylation was detected by Western Blot. The localization of Paxillin was determined by co-immunofluorescence staining after transfection with cyanine fluorescence protein tagged GIT1RNAh (CFP-GIT1-RNAh)(experimental group) and GFP-RNAh (CFP-GFP-RNAh)(control group). The role of GIT1-RNAh (experimental group) and GFP-RNAh (control group) adenovirus in osteoblasts migration was determined by wound healing assay. Results Immunofluorescence staining results showed that the GIT1-RNAh significantly inhibited endogenous GIT1 expression, interfered Paxillin distribution.Western Blot results showed that Paxillin phosporylation was obviously inhibited in osteoblasts infected with GIT1-RNAh adenovirus (P＜0.05). The wound healing assay results howed that GIT1-RNAh adenovirus significantly inhibited osteoblast migration induced by PDGF. Conclusion GIT1-RNAh inhibits osteoblasts migration by interfering paxillin distribution and decrease Paxillin phosphorylation.
Objective To explore the effects of Zhaoke defibrase and anti alpha;vbeta;3mAb (23C6) on the adhesion and immigration of bovine retinal vascular endothelial cells. Methods The culture dishes coated with vitronectin (Vn) and collagen，assays of adhesion and immigration were performed 60 minutes after different concentration of Zhaoke defibrase and anti-alpha;vbeta;3 mAb was added to the bovine retinal vascular endothelial cells. The apoptosis of bovine retinal vascular endothelial cells induced by Zhaoke defibrase and anti-alpha;vbeta;3 mAb was detected by electron microscopy. Results Both Zhaoke defibrase and anti-alpha;vbeta;3 mAb inhibited the adhesion and immigration of bovine retinal vascular endothelial cells in a dose-dependent manner. The inhibited concentration (IC50) of Zhaoke defibrase was less than 0.05 mu;mol/L, while (IC50) of anti-alpha;vbeta;3 mAb was more than 2.5 mu;mol/L. 81.8% endothelial cells adhering to Vn were inhibited by 0.1 mu;mol/L Zhaoke defibrase, while 76.3% by endothelial cells adhering to Vn were inhibited by 10 mu;mol/L anti-alpha;vbeta;3 mAb. Typical apoptosis cells were found in bovine retinal vascular endothelial cells after affected by Zhaoke defibrase and anti-alpha;vbeta;3 mAb. Conclusion Both Zhaoke defibrase and anti- alpha;vbeta;3mAb can significantly inhibit the adhesion and immigration of bovine retinal vascular endothelial cells to extracellular matrix， and the mechanism may lie in inducing the apoptosis of endothelial cells. (Chin J Ocul Fundus Dis, 2005,21:118-121)
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 reduce the incidence of peritoneal dialysis (PD) catheter complications through a continuous quality improvement (CQI) process. MethodsTwenty-nine patients with catheters inserted (from January 2011 to March 2011) before CQI, and another 41 patients with catheters inserted (between April 2011 and January 2012) after CQI were observed and analyzed. The possible causes of complications of catheter were summarized, and then on the basis of that, a PDCA four-step (plan-do-check-act) method was designed with a view to reducing the incidence of postoperative complications. ResultsPD catheter dysfunction decreased from 6.90% to 2.44%. The incidence of leakage decreased from 44.83% to 9.76%. ConclusionCQI is a useful method to reduce the incidence of postoperative complications of PD catheter in peritoneal dialysis.
Low shear stress is a component of the tumor microenvironment in vivo and plays a key role in regulating cancer cell migration and invasion. The integrin, as a mechano-sensors mediating and integrating mechanical and chemical signals, induce the adhesion between cells and extracellular matrix (ECM). The purpose of this study is to investigate the effect of low shear stress(1.4 dyn/cm2)on the migration of HepG2 cells and the expression of integrin. Scratch wound migration assay was performed to examine the effect of low shear stress on the migration of HepG2 cells at 0 h, 1 h, 2 h and 4 h, respectively. F-actin staining was used to detect the expression of F-actin in HepG2 cells treated with low shear stress at 2 h and 4 h. Western blot analysis was carried out to determine the effect of low shear stress on the expression of integrin at different durations. The results showed that the migrated distance of HepG2 cells and the expression of F-actin increased significantly compared with the controls. The integrin α subunits showed a different time-dependent expression, suggesting that various subunits of integrin exhibit different effects in low shear stress regulating cancer cells migration.