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

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

Until recently, vascular endothelial growth factor (VEGF) was the only growth factor proven to be specific and critical for blood vessel formation. Other long-known factors, such as the fibroblast growth factors (FGFs), platelet-derived growth factor, or transforming growth factor-beta, had profound effects in endothelial cells. But such factors were nonspecific, in that they could act on many other cells, and it seemed unlikely that these growth factors would be effective targets for treatment of endothelial cell diseases. A recently discovered endothelial cell specific growth factor, angiopoietin, has greatly contributed to our understanding of the development, physiology, and pathology of endothelial cells (Davis et al., 1996; Yancopoulos et al., 2000). The recent studies that identified and characterized the physiological and pathological roles of angiopoietin have allowed us to widen and deepen our knowledge about blood vessel formation and vascular endothelial function. Therefore, in this review, we describe the biomedical significance of these endothelial cell growth factors, the angiopoietins, in the vascular system under normal and pathological states.
Alternative Splicing ; Angiogenesis Factor/genetics/*metabolism ; Animal ; Cell Survival ; Endothelial Growth Factors/metabolism ; Endothelium, Vascular/cytology/*physiology ; Hematopoiesis/physiology ; Human ; Intercellular Signaling Peptides and Proteins/metabolism ; Lymphokines/metabolism ; Membrane Glycoproteins/genetics/*metabolism ; Neoplasm Proteins/metabolism ; Neovascularization, Pathologic ; Neovascularization, Physiologic ; Signal Transduction/physiology ; Urogenital System/physiology

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

OBJECTIVEGene therapy has been becoming one of the most attractive medical areas. But the using of gene therapy in plastic surgery is relatively scarce. Our purpose was to investigate the effect of naked plasmid encoding Vascular Endothelial Growth Factor on the viability of the random skin flap by directly injected subcutaneously.

METHODS30 female Sprague-Dawley rats randomly divided into three groups. A random dorsal skin flap of 3 cm x 9 cm was elevated in each of the rats. And 1 ml double-distilled water solution was injected subcutaneously, which was only water in group 1 during the operation, 200 micrograms VEGF cDNA plasmid in group 2 during the operation, 200 micrograms pcDNA3.1/zeo(+)--VEGF in group 3, 24 hours before the operation, respectively. 7 days after the operation, all the animals were sacrificed by overdose anesthetic. The survival tissue was measured with planimetry. Two samples were harvested from each group for pathological check and immunohistochemical test.

RESULTSImmunohistochemical staining demonstrated that there was human VEGF deposited around the capillary in the flaps treated with VEGF gene. The flaps treated with VEGF gene had a larger percentage of survival skin (group 1 = 47% +/- 5.4%, group 2 = 65.4% +/- 6.3%, group 3 = 72.3% +/- 8.5%, P < 0.05).

CONCLUSIONVEGF gene directly injected into subcutaneous can express VEGF. It makes the gene therapy simple and practical and will be promising future in the tissue transplantation.

Animals ; Endothelial Growth Factors ; genetics ; Female ; Genetic Therapy ; Injections, Subcutaneous ; Lymphokines ; genetics ; Rats ; Rats, Sprague-Dawley ; Surgical Flaps ; physiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the pro-angiogenic effects of several multiple myeloma (MM) cell line culture supernatants on human bone marrow endothelial cell (HBMEC) proliferation, migration, and capillary formation, and the anti-angiogenic effects of thalidomide.

METHODSHBMEC was cultured in the presence of MM cell lines (IM9, XG1, U266 and MOLP-5) supernatants. Proliferation and migration of HBMEC were determined, capillary-like tubule formation of HBMEC was examined in fibrin and Matrigel. The inhibiting effect of thalidomide was investigated by adding it into myeloma cell line culture supernatants. Vascular endothelial growth factor (VEGF) was measured by ELISA.

RESULTS(1) MM cell lines culture supernatants promoted HBMEC proliferation and migration. (2) In fibrin and Matrigel, capillary-like tubule network formation promoted by the supernatants. (3) All of these effects could be inhibited by thalidomide. (4) This effect was not related to VEGF in the supernatants.

CONCLUSIONSMM cell line promote proliferation, migration and tubule formation by secreting VEGF or other several cytokines. Thalidomide can inhibit these effects.

Angiogenesis Inhibitors ; pharmacology ; Bone Marrow ; blood supply ; Cell Division ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Endothelial Growth Factors ; metabolism ; Endothelium, Vascular ; cytology ; drug effects ; physiology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Lymphokines ; metabolism ; Multiple Myeloma ; pathology ; secretion ; Neovascularization, Physiologic ; drug effects ; Thalidomide ; pharmacology ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the effect of antisense vascular endothelial growth factor (VEGF)(121) cDNA transfection on the growth of K562 cells in nude mice.

METHODSK562 cells transfected with the antisense (AS) or sense (S) VEGF(121) cDNA, and the vector (V, pcDNA3) alone were transplanted subcutaneously into nude mice and the growth of the transfected cells in vivo was investigated. The effects of transfected K562 cells on human bone marrow endothelial cells (BMEC) were analyzed by MTT assay, the microvessel density (MVD) in tumor mass by vWF immunohistochemistry stain.

RESULTSK562/V tumor grew more slowly [(207.5 +/- 192.9) mm(3) vs (445.0 +/- 150.9) mm(3), P < 0.05] and K562/S tumor more rapidly than K562/V tumor did [(1 174.6 +/- 508.7)/mm(3) vs (445.0 +/- 150.9) mm(3), P < 0.01]. K562/S cell culture supernatant was more strongly in promoting the proliferation of BMEC than K562/V supernatant did, but K562/AS supernatant resulted in a marked decrease of the promoting effect as compared with K562/V's. The MVDs in K562/AS, K562/S, and K562/V tumors were [(11.0 +/- 7.6)/0.72 mm(2) vs (50.8 +/- 11.7)/0.72 mm(2) vs (18.9 +/- 7.0)/0.72 mm(2)], respectively.

CONCLUSIONSAntisense VEGF(121) cDNA transfected K562 cells show growth retardation in transplanted nude mice, decrease of tumor MVD, and decrease of promoting BMEC proliferation capacity.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Division ; genetics ; physiology ; Culture Media, Conditioned ; pharmacology ; DNA, Antisense ; genetics ; DNA, Complementary ; genetics ; Endothelial Growth Factors ; genetics ; physiology ; Endothelium, Vascular ; cytology ; drug effects ; Female ; Humans ; K562 Cells ; Lymphokines ; genetics ; physiology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; blood supply ; genetics ; pathology ; Neovascularization, Pathologic ; genetics ; physiopathology ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

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