OBJECTIVE: To investigate the biological effect of medical recombinant human-derived collagen functional dressings in wound healing. METHODS: MTT assay and RTCA assay were used to detect cell toxicity and proliferation. Scratch assay and Transwell cell migration assay were used to detect cell motility and migration ability. Enzyme-linked immunosorbent assay was used to detect the contents of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-endothelial cell adhesion molecule (CD31) in the supernatant of four types of cells. After animal surgery, the surgical wound was taken at 1 week, 4 weeks and 13 weeks, respectively, for hematoxylin eosin (HE) staining and immunohistochemistry to observe the inflammatory response and CD31 expression of the wound. RESULTS: Medical recombinant human-derived collagen functional dressing promotes cell proliferation and migration, enhances wound angiogenesis by upregulating the expression of VEGF, FGF, and CD31 in human dermal vascular endothelial cells (HDVEC) and human vascular endothelial cells (HVEC), thereby improving local blood supply to the wound, regulating the inflammatory response of the wound, and accelerating wound healing. CONCLUSION Recombinant type Ⅲ humanized collagen plays an important role in wound healing.
Humans ; Wound Healing/drug effects* ; Recombinant Proteins/pharmacology* ; Animals ; Cell Proliferation ; Cell Movement ; Collagen/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Bandages ; Platelet Endothelial Cell Adhesion Molecule-1/metabolism* ; Endothelial Cells ; Fibroblast Growth Factors/metabolism*

Neamine, a non-toxic derivative of neomycin, has recently been shown to have antitumor activities in various types of cancers. However, its effect on pancreatic cancer is still unknown. The study aimed to investigate its antitumor activity on pancreatic cancer and the underlying mechanisms. MTT assay was used to observe the effect of neamine on angiogenin (ANG)-induced AsPC-1 cell proliferation. Tissue microassay and immunofluorescence staining were used to detect the expression of ANG and its nuclear translocation, respectively. Tumor xenografts were established by subcutaneous inoculation of AsPC-1 pancreatic cancer cells into the right flanks of nude mice, and neamine was injected subcutaneously. Immunohistochemistry was done to observe the expression of ANG, CD31 and Ki-67 in tumor xenografts. It was found that neamine blocked the nuclear translocation of ANG effectively and inhibited ANG-induced AsPC-1 cell proliferation in a dose-dependent manner. Neamine had anti-tumor effects on AsPC-1 xenograft models. Consistently, neamine reduced the expression levels of ANG, Ki-67 and CD31 in tumor xenografts. It was concluded that neamine may be a promising agent for treatment of pancreatic cancer.
Adult ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; therapeutic use ; Carcinoma ; drug therapy ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Framycetin ; pharmacology ; therapeutic use ; Humans ; Ki-67 Antigen ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Pancreatic Neoplasms ; drug therapy ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; Ribonuclease, Pancreatic ; genetics ; metabolism

OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

OBJECTIVETo explore the protein expression of heme oxygenase-1 (HO-1) and platelet endothelial cell adhesion molecules-1 (PECAM-1) in human coronary artery endothelial cells induced with Zinc Oxide Nanoparticle (ZnO-NPs).

METHODSMTT assay was used to determine the cell viability of ZnO-NPs. Levels of HO-1 and PECAM-1 protein in culture supernatants were measured using ELISA after human coronary artery endothelial cells were treated with different concentrations (0, 10, 20, 40µg/ml) of ZnO-NPs for 24 h.

RESULTSThe cell viability of human coronary artery endothelial cells in each group was 89.76%, 83.61%, 63.10%, 53.20%, 48.11%, 42.35%, 38.06%, 25.44% respectively when treated with different concentrations of ZnO-NPs (12.5, 25, 50, 70, 80, 90, 100, 200µg/ml). Protein levels of HO-1 (ng/L) in each group were 0.041±0.011, 0.512±0.076, 0.906±0.059, 1.062±0.089 respectively after the stimulation of different concentrations of ZnO-NPs (0, 10, 20, 40µg/ml). Comparisons in each group were statistically significant (P < 0.05). Protein levels of PECAM-1 (µg/L) in each group were 7.966 ± 0.046, 7.993 ± 0.036, 8.629 ± 0.052, 8.811 ± 0.039 respectively after the stimulation of different concentrations of ZnO-NPs (0, 10, 20, 40 µg/ml). Compared with the control group, protein levels of PECAM-1 increased (P < 0.05) when the concentration of ZnO-NPs was 20µg/ml or 40 µg/ml.

CONCLUSIONZnO-NPs stimulation could inhibit the viability of human coronary artery endothelial cells and upregulate the protein expression of HO-1 and PECAM-1.

Blood Platelets ; Cell Survival ; Coronary Vessels ; Endothelial Cells ; drug effects ; Heme Oxygenase-1 ; metabolism ; Humans ; Nanoparticles ; toxicity ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Zinc Oxide ; toxicity

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.
Animals ; Disease Models, Animal ; GPI-Linked Proteins ; genetics ; Gene Expression Regulation ; Genetic Therapy ; Humans ; Immunity, Innate ; genetics ; Inflammation ; genetics ; microbiology ; pathology ; Isoantigens ; genetics ; Mice ; Mice, Knockout ; Neutrophils ; metabolism ; pathology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Receptors, Cell Surface ; genetics ; Staphylococcus aureus ; pathogenicity ; Transcriptional Activation

BACKGROUNDThe clinical applications of fibrin glue span over several surgical modalities. The aim of this study was to evaluate the biocompatibility and biodegradation of different formulations of platelet-rich fibrin glue in vivo and examine its effects on the neovascularization of wound sites.

METHODSHuman-derived single-unit fibrin glue was prepared. Incisions were made on the backs of rats, and these were coated with homemade glues containing different concentrations of aminomethylbenzoic acid (Groups A-F) or commercial adhesives (Group G). A sham control group was included (Group H). The wounds were examined by histological analysis and immunohistochemistry at several time points.

RESULTSSuccessful wound closure was achieved in all groups by day 12. Acute inflammation occurred during the first six days, but gradually disappeared. The longest sealant duration was achieved using the lowest concentration of anti-fibrinolytic agent in a 1:10 volume ratio with cryoprecipitate. Expression levels of the platelet endothelial cell adhesion molecule-1 were significantly higher in Groups A and C compared to the control groups (Groups G and H) on day 3 (P < 0.05).

CONCLUSIONSSingle-unit platelet-rich fibrin glue has excellent biocompatibility and is associated with the upregulation of neovascularization. The addition of aminomethylbenzoic acid could prevent the degradation of fibrin glue.

Animals ; Female ; Fibrin Tissue Adhesive ; adverse effects ; therapeutic use ; Humans ; Immunohistochemistry ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Wound Healing ; drug effects

BACKGROUNDConsidering the existence of a large number of liver cell degeneration and necrosis in fibrotic liver, liver function was damaged severely and could not effectively regenerate after partial hepatectomy (PHx). The aim of this study was to investigate whether decorin (DCN) could promote the liver regeneration after PHx in fibrotic mice.

METHODSForty mice (5-week-old, Balb/c) were injected with CCl4 intraperitoneally and liver fibrosis model was established after 5 weeks. The survival mice were randomly divided into two groups: control group and DCN group. Then, we performed 70% PHx on all these mice and injected DCN or phosphate-buffered saline plus normal saline (NS) to each group, respectively, after surgery. Liver body weight ratio (LBR), quantitative real-time polymerase chain reaction, and immunohistochemistry were used to analyze liver regeneration and fibrosis degree in both groups, and to find out whether exogenous protein DCN could promote the regeneration of fibrosis liver after PHx.

RESULTSExpressions of a-smooth muscle actin (SMA) mRNA and LBR had significant increases in the DCN group at postoperative Day 3 (POD 3, P < 0.05). The protein expressions of CD31, a-SMA, and tumor necrosis factor (TNF)-a were higher in the DCN group than those in the control group by immunohistochemistry at POD 3 (P < 0.05).

CONCLUSIONExogenous protein DCN could promote liver regeneration after PHx in fibrotic mice.

Animals ; Decorin ; therapeutic use ; Hepatectomy ; Immunohistochemistry ; Liver Cirrhosis ; drug therapy ; metabolism ; surgery ; Liver Regeneration ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo explore the combined anti-tumor effect of radiation therapy and gene-targeted suppression of tumor neovasculature in lung adenocarcinoma in vivo, and to explore the feasibility of micro-PET/CT in dynamic evaluation of treatment effectiveness.

METHODSThirty 5-6-week old male BALB/c nude mice were used in this study. The mouse models of xenotransplanted human lung adenocarcinoma were divided into 5 groups at random, six mice in each group: the control group, radiation treatment alone group and three groups of recombinant baculovirus plus radiation treatment (intratumoral injection, tail vein injection, and intramuscular injection). The tumor volume was measured every 2 days. Growth delay time (GD) and growth inhibition rate was calculated. FDG metabolism was evaluated by micro-PET-CT before and after treatment. The expressions of VEGF, CD31 and Ki-67 were detected by immunohistochemistry (IHC).

RESULTSThe tumor growth delay was >12 days, and the tumor inhibition rate was >45% in the recombinant baculovirus combined with radiotherapy groups, significantly higher than that of the radiotherapy alone group (P < 0.05). Immunohistochemical analysis showed that the expressions of VEGF, CD31 and Ki-67 were significantly lower than that in other groups (P < 0.05). The micro-PET-CT assessment showed that the FDG-metabolism in the recombinant baculovirus combined with radiotherapy groups was significantly reduced (P < 0.05), and the SUVmax (FDG metabolism) of transplanted tumors after treatment was also markedly decreased in comparison with that of the control group. The tumor volume after treatment was significantly correlated with SUVmax in the recombinant baculovirus intratumoral injection + radiotherapy group(r = 0.976), recombinant baculovirus intravenous injection + radiotherapy group (r = 0.954), recombinant baculovirus intramuscular injection + radiotherapy group (r = 0.929), and radiotherapy alone group (r = 0.871, P < 0.05).

CONCLUSIONSThe recombinant baculovirus containing Egr1 promoter and K5 gene combined with radiotherapy enhances the suppressing effect on the growth of lung adenocarcinoma in the tumor-bearing nude mice. The inducibility of Egr1 promoter by radiation allows the targeting and controllability of treatment. Micro-PET-CT results have a good correlation with the treatment effectiveness. Therefore, it can be used in real-time evaluation of tumor metabolic function in vivo.

Adenocarcinoma ; metabolism ; pathology ; radiotherapy ; Animals ; Baculoviridae ; genetics ; Cell Line, Tumor ; Combined Modality Therapy ; Early Growth Response Protein 1 ; genetics ; physiology ; Fluorodeoxyglucose F18 ; Genetic Therapy ; Genetic Vectors ; Humans ; Ki-67 Antigen ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; radiotherapy ; Male ; Mice, Inbred BALB C ; Mice, Nude ; Molecular Targeted Therapy ; Neoplasm Transplantation ; Peptide Fragments ; genetics ; physiology ; Plasminogen ; genetics ; physiology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Positron-Emission Tomography ; Promoter Regions, Genetic ; Random Allocation ; Recombinant Proteins ; genetics ; Tomography, X-Ray Computed ; Tumor Burden ; Vascular Endothelial Growth Factor A ; metabolism

To explore the feasibility of mesenchymal stem cells (MSCs) acting as seed cells in tissue engineering, we isolated human bone marrow MSCs and differentiated them into vascular endothelial-like cells (ELCs) in vitro. Bone marrow mononuclear cells (BMSCs) were isolated by the method of percoll density centrifugation, and seeded in Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum. MSCs were purified through multiple adherent cultures, and differentiated into ELCs induced by endothelial cell growth medium-2 (EBM-2) medium containing vascular endothelial growth factor (VEGF), human fibroblast growth factor (hFGF), insulin like growth factors 1 (IGF-1), and human epidermal growth factor (hEGF). The relative biologic characteristics of ELCs including cell morphology and phenotype were studied by inverted microscope and flow cytometry. The induced cells were identified by immunofluorescence with CD31 and Von Willebrand factor (vWF). The results showed that the morphology of MSCs was long-spindle and vortex-like growth. After induction of differentiation, the cells were round, and similar to vascular endothelial cells (ECs). Flow cytometric analysis revealed that ELCs expressed ECs specific surface markers of CD31 and vascular endothelial cadherin (VE-cadherin), but not CD133. Immunofluorescence results also confirmed that ELCs expressed CD31 and vWF. The results suggested that ELCs possed similar cell biological characteristics with ECs. In one word, human MSCs derived from bone marrow have the potential to differentiate into ECs in vitro, and show clinical feasibility acting as ideal donor cells of vascular tissue engineering.
Antigens, CD ; metabolism ; Bone Marrow Cells ; Cadherins ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Culture Media ; chemistry ; Endothelial Cells ; cytology ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factors ; pharmacology ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; pharmacology ; von Willebrand Factor ; metabolism

OBJECTIVETo investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation.

METHODSDPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction.

RESULTSThe relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P < 0.05). For in vitro tube formation assay, tubular structures were formed on the matrigel by differentiated DPC. A total of 47 miRNA were differentially expressed, in which 15 miRNA were up-regulated and 32 miRNAs down-regulated in differentiated DPC compared with the control. Of these, 4 miRNA were confirmed by qRT-PCR. The target genes of differential miRNA were predicted to associate with several biological functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway.

CONCLUSIONSThe differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

Antigens, CD ; Cadherins ; Cell Differentiation ; Collagen ; Computational Biology ; Dental Pulp ; metabolism ; Drug Combinations ; Fibroblast Growth Factor 2 ; Humans ; Laminin ; MicroRNAs ; Platelet Endothelial Cell Adhesion Molecule-1 ; biosynthesis ; Proteoglycans ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Vascular Endothelial Growth Factor A ; Wnt Signaling Pathway ; von Willebrand Factor