Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
Animals ; Antigens, CD34 ; immunology ; Binding Sites ; genetics ; CHO Cells ; Cell Division ; drug effects ; physiology ; Colony-Forming Units Assay ; Cricetinae ; Endothelial Growth Factors ; pharmacology ; Fetal Blood ; cytology ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Glycoproteins ; genetics ; pharmacology ; physiology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Interleukin-3 ; pharmacology ; Lymphokines ; pharmacology ; Membrane Proteins ; genetics ; RNA ; genetics ; metabolism ; Receptor, Notch1 ; Receptors, Cell Surface ; Recombinant Proteins ; isolation & purification ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; Transcription Factors ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the response of nasal mucosa epithelial cells to hypoxia in terms of formation of nasal polyps (NP).

METHODSEpithelial cells of NP and inferior turbinate (IT) were cultured serum-free under normal oxygen and hypoxic circumstances with stimulation of IL-1 beta and TNF alpha. The vascular endothelial growth factor (VEGF) mRNA and VEGF protein levels of the cultured cells were detected using in situ hybridization and ELISA, respectively.

RESULTSThe expression of VEGF mRNA was significantly higher in epithelial cells of NP than in IT exposed to pro-inflammatory cytokines or hypoxia (P < 0.01). VEGF levels were higher in NP epithelial cells than those of IT (P < 0.01) under hypoxia.

CONCLUSIONVEGF-induced by hypoxia is very important for the early stages of forming polyps.

Cell Hypoxia ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Interleukin-1 ; pharmacology ; Lymphokines ; genetics ; Nasal Mucosa ; metabolism ; Nasal Polyps ; etiology ; metabolism ; RNA, Messenger ; analysis ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

To investigate the relationship of the expression of vascular endothelial growth factor (VEGF) and C-myc with the occurrence, development and metastasis of gallbladder carcinoma, the expression levels of VEGF and C-myc in gallbladder carcinoma tissue (n = 30) and in normal gallbladder tissue (n = 20) were examined by immunochemistry. Results show that the positive rates of VEGF and C-myc in gallbladder carcinoma tissue were 80% and 63.3% respectively, and 45% and 25% respectively in normal gallbladder tissue. The positive rates of VEGF and C-myc were significantly higher in gallbladder carcinoma than in normal gallbladder tissue. The expression of VEGF and C-myc in gallbladder carcinoma related to the metastasis of gallbladder carcinoma. VEGF and C-myc play important roles in the occurrence, development and metastasis of gallbladder carcinoma.
Adult ; Aged ; Endothelial Growth Factors ; biosynthesis ; physiology ; Female ; Gallbladder Neoplasms ; metabolism ; pathology ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; physiology ; Lymphokines ; biosynthesis ; physiology ; Male ; Middle Aged ; Neoplasm Metastasis ; Proto-Oncogene Proteins c-myc ; biosynthesis ; physiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODSThe HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTSHFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONHFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Cell Adhesion ; Cell Cycle ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; metabolism ; Epidermis ; Fetus ; G1 Phase ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Keratinocytes ; cytology ; Keratins ; analysis ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Microscopy, Fluorescence ; Plasmids ; genetics ; Stem Cells ; chemistry ; cytology ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Angiogenesis is a necessary step in tumor progression, and it correlates an unfavorable prognosis. In multiple myeloma, bone marrow microvessel density and angiogenesis grading correlated with plasma cell labeling index and are poor survival predictors, but the study of myeloma's angiogenesis is very rare. This article was to study the effect of multiple myeloma cell line conditioned media on the proliferation, migration and angiogenesis of human bone marrow endothelial cells (HBMEC). The multiple myeloma cell line conditioned media were obtained by using RPMI 1640 media containing 2% fetal bovine serum (FBS) to cultivate myeloma cell lines for 18 hours. Proliferation and migration of HBMEC were detected by using those media to cultivate HBMEC. Capillary tube formation was performed by using microcarriers cytodex-3 covered with HBMEC in three-dimensional fibrin matrices. The results showed that myeloma conditioned media induced HBMEC's proliferation and migration (P < 0.001), and those media induced capillary tube formation (length and width) of HBMEC (P < 0.001). It was concluded that myeloma cell lines induce HBMEC's proliferation, migration, and capillary tube formation by secreting several cytokines.
Bone Marrow Cells ; cytology ; Cell Division ; Cell Movement ; Endothelial Growth Factors ; analysis ; physiology ; Humans ; Intercellular Signaling Peptides and Proteins ; analysis ; physiology ; Lymphokines ; analysis ; physiology ; Multiple Myeloma ; blood supply ; chemistry ; pathology ; Neovascularization, Pathologic ; etiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

The present study identified the favorable environment conditions for Spermatogomial stem cells in vitro according to their unique biological properties. Three growth factors, stem cell factor (SCF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) were all found to independently contribute to the proliferation of mouse spermatogonial stem cell. The percentage of cell proliferation significantly enhanced by SCF at 30 ng/mL but decreased with heightening its combination after cultured 120 hours. The mice spermatogonial stem cells were significantly proliferated after 120 hours' culture with 10 ng/mL and 20 ng/mL (P < 0.01) of LIF, between 20 ng/mL and 50 ng/mL (P < 0.01) for bFGF. SCF and bFGF were significantly enhanced mice spermatogonial stem cells proliferation after these three factors combination. For LIF, no obvious effect was observed.
Animals ; Cell Division ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; pharmacology ; Growth Inhibitors ; pharmacology ; Interleukin-6 ; Leukemia Inhibitory Factor ; Lymphokines ; pharmacology ; Male ; Mice ; Spermatogonia ; drug effects ; physiology ; Stem Cell Factor ; pharmacology ; Stem Cells ; drug effects ; physiology

OBJECTIVETo investigate the killing effect of herpes simplex virus thymidine kinase gene expression actuated by the promoter of the vascular endothelial growth factor gene on human hepatocellular tumor cells under hypoxic condition.

METHODSRecombinant adenoviral vectors, AdVEGF-tk and AdVEGF-GFP, were constructed with HSV-tk or GFP under the control of VEGF promoter through AdEasy system. Then GFP expression in hepatoma cell line HepG2 and normal liver cell line L02 transfected with AdVEGF-GFP were observed under fluorescence microscope, and the sensitivity to GCV of the AdVEGF-tk-transfected cells under normoxia or hypoxia condition were monitored by MTT method.

RESULTSGFP expression actuated by VEGF promoter was detected in sporadic L02 cells, but in almost all HepG2 cells after transfected with AdVEGF-GFP. With GCV at 10 micro g/ml and MOI at 100, L02 cells were insensitive to GCV under oxic condition, but more than 70% L02 cells were killed under hypoxic condition. Moreover, HepG2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under hypoxia (over 80% killed) as compared with normoxia (over 60% killed) conditions.

CONCLUSIONHypoxia enhances the GCV sensitivity of human hepatocellular tumor cells infected with recombinant AdVEGF-tk under the control of VEGF promoter.

Adenoviridae ; genetics ; Carcinoma, Hepatocellular ; pathology ; Endothelial Growth Factors ; genetics ; Gene Expression ; drug effects ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Hypoxia ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver Neoplasms ; pathology ; Lymphokines ; genetics ; Oxygen ; pharmacology ; Promoter Regions, Genetic ; physiology ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate whether direct administration of adenoviral vectors (Ad) containing the complementary deoxyribonucleic acid (cDNA) of vascular endothelial growth factor 165 (Ad-VEGF165) induces porcine coronary collateral vessel formation, improves regional myocardial perfusion and function and is safe.

METHODSThree weeks after miniature swine underwent left thoracotomy and placement of an Ameroid constrictor on the left circumflex coronary artery (LCX), Ad-VEGF165 (n = 6) or the control, Ad expressing beta-galactosidase cDNA (Ad-Gal, n = 6), was directly administered into the ischemic myocardium in the circumflex distribution. All animals were sacrificed 4 wk after the second surgery. Myocardial perfusion and function were assessed by electrocardiogram-gated single photon emission computed tomography (GSPECT) imaging. Ex vivo coronary angiography was performed to examine collateral vessels. Toxicity was assessed by blood analyses on the day just before (day 0) and on day 1, 3, 7, 28 after vector delivery and by vascular, myocardial and liver histology after sacrifice.

RESULTSGSPECT imaging 4 wk after administration of Ad-VEGF165 demonstrated significant reduction in ischemic area (P < 0.01) and rest ischemic severity (P < 0.01) and significant improvement in the left ventricular ejection fraction (P < 0.01) and regional wall motion (P < 0.05) compared with that of Ad-Gal and before administration of Ad-VEGF165. Collateral vessel development assessed by coronary angiography was significantly greater in the Ad-VEGF165 group than in the Ad-Gal group (P < 0.05). General safety parameters, including routine blood parameters, liver and kidney function and cardiac specific parameters demonstrated no difference between Ad-VEGF165 and Ad-Gal animals except for the red blood cell count on day 28 (P < 0.05) and blood urea nitrogen on day 7 (P < 0.05). Only transient elevations in creatine phosphokinase (P < 0.05) and aspartate transaminase (P < 0.05) on day 1 were revealed compared with that before vector administration in both groups. Histologically, no atherosclerotic lesion in the circumflex and no inflammation in liver were revealed and only a small myocardial necrosis was observed in one Ad-VEGF165 animal (area < or = 20%) and one Ad-Gal animal (area < 10%).

CONCLUSIONSAd-VEGF165 can induce coronary collateral vessel formation, improve regional myocardial perfusion and function and is safe by means of direct injection, which suggesting that this strategy may be useful in treating human ischemic heart disease.

Adenoviridae ; genetics ; Animals ; Collateral Circulation ; Coronary Angiography ; Coronary Vessels ; physiopathology ; DNA, Complementary ; administration & dosage ; genetics ; Electrocardiography ; Endothelial Growth Factors ; genetics ; physiology ; Female ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; genetics ; Lymphokines ; genetics ; physiology ; Male ; Myocardial Ischemia ; diagnostic imaging ; genetics ; therapy ; Neovascularization, Physiologic ; physiology ; Swine ; Swine, Miniature ; Tomography, Emission-Computed, Single-Photon ; methods ; Treatment Outcome ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the feasibility of endothelialization of bioprosthesis by transfer of vascular endothelial growth factor (VEGF) gene.

METHODSBovine pericardium treated with glutaraldehyde and L-glutamic acid was positioned into the pig right atrium. pcD(2)/hVEGF(121) gene (1 mg) was transferred into the right ventricular myocardium using surgical sutures Reverse transcri ption polymerase chain reaction (RT PCR) was employed to evaluate the expression of myocardial VEGF mRNA. The determination of concentrations of VEGF protein in blood from both the right atrium and peripheral vein, and histological and ultrastructural analysis of implanted bovine pericardium were completed simultaneously.

RESULTSThe concentration of VEGF derived from the right atrium in pcD(2)/hVEGF(121) group was significantly higher than that in the pcD(2) group 10 days after VEGF gene transfer (P < 0.01). The expression of myocardial VEGF mRNA in pcD(2)/hVEGF(121) group was much higher in comparison with that in the pcD(2) group. The morphological analysis demonstrated that the coverage rate of host endothelium in the pcD(2)/hVEGF(121) group was 2.6 times as fast as that in the pcD(2) group at 16 days after VEGF(121) gene transfer (P < 0.01). Entire endothelialization occurred at 30 days after VEGF gene transfer. In addition, higher expression of myocardial VEGF mRNA was still available.

CONCLUSIONSVEGF gene transfer by surgical suture can remarkably accelerate endothelialization of bioprosthesis, which may provide a new approach for inhibiting biological valve calcification and improve biocompatibility and long-term durability of the bioprosthesis.

Animals ; Bioprosthesis ; Endothelial Growth Factors ; analysis ; genetics ; Endothelium, Vascular ; physiology ; Female ; Gene Transfer Techniques ; Heart Valve Prosthesis ; Humans ; Lymphokines ; analysis ; genetics ; Male ; RNA, Messenger ; analysis ; Swine ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the value of application of recombinant human VEGF to accelerate flap viability in a rat model of non-ischemic prefabricated flap.

METHODSPrefabricated Flaps were created in 48 SD rats. An autologous tail artery loop was anastomosed to the femoral artery and vein, and implanted subcutaneously in the lower abdomen. Flaps were divided into four groups of 12 each. At the time of loop implantation, the control groups received 0.9% NaCl (Control 1) and 16% (V/W) polyvinyl alcohol (PVA) solution (Control 2). The treatment groups received VEGF in 0.9% NaCl (treatment 1) and VEGF in PVA (treatment 2). In each group, a 3 cm x 4 cm flap nurtured by the tail artery pedicle was elevated and resutured into place after 3, 4 and 5 weeks. The percentage of surviving skin of each flap was determined by planimetry 7 days after flap elevation.

RESULTSMean skin survival areas at 3, 4, and 5 weeks were 1%, 0%, 10% in control; 0%, 16%, 25% in control 2; 3.57%, 39.13%, 75.00% in treatment 1; 8.13%, 41.98%, 58.41% in treatment 2. VEGF significantly improved flap survival by 5 weeks (P < 0.05).

CONCLUSIONThese results suggest VEGF can accelerate maturation of prefabricated flaps.

Animals ; Endothelial Growth Factors ; pharmacology ; Female ; Lymphokines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Surgical Flaps ; physiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors