Primary HLSCs were successfully cultured and assayed by AE5 in vitro. Constructed eukaryotic expressive vector of pEGFP-bFGF was transferred into the human limbal stem cells by the liposome-mediated technique, and 48 hours later, specific green fluorescence was observed by fluorescence microscope. The gene transfeetion efficiency was 20%-30%. Then the model of cells injury was created by use of NaOH. The cells were divided into four groups: Normal, bFGF, NaOH and bFGF+NaOH. The cellular viability in each group was measured by MTT colorimetry, and the cellular apoptosis rate and necrosis rate were observed by laser scanning confocal microscopy. The cellular viability in bFGF+NaOH group was higher than that in NaOH group (P < 0.05) ,while the cellular apoptosis rate plus necrosis rate displayed significant difference between the two groups (P < 0.05). The pEGFP-bbGF gene was noted to be successfully transferred into HLSCs and the cells were found growing well. These indicated that bFGF gene has a protective effect on the HLSCs injured by NaOH. We have also probed the feasibility of trying the treatment for ocular surface disease through gene engineering recombined tissue engineering.
Cells, Cultured ; Cornea ; cytology ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Gene Expression ; Genetic Therapy ; methods ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cells ; cytology ; Tissue Engineering ; Transfection ; Vascular Endothelial Growth Factors ; genetics ; metabolism

OBJECTIVETo study the relationship of TCM syndrome type of gastric mucosal epithelial growth factor (EGF), vascular endothelial growth factor (VEGF) and proliferative cell nuclear antigen (PCNA) in patients with chronic atrophic gastritis (CAG) for exploring the essence of TCM type and providing a theoretical basis of clinical treatment.

METHODSTCM syndrome type of 200 patients with diagnosis of CAG confirmed by fibro-gastroscope and pathological examination were differentially classified, and the expressions of EGF, VEGF and PCNA in different types were determined using immunohistochemistry.

RESULTSPatients were differentiated as Pi-Wei deficiency type (Type I ) in 72; Gan-Wei disharmony type (Type II ) in 43; Pi-deficiency with qi stagnation type (Type III) in 32; Wei-yin deficiency type (Type IV) in 24; Pi-Wei damp-heat type (Type V) in 14; and Wei-collateral stasis obstruction type (Type VI) in 5. The difference of PCNA expression level between Type II with Type I , III and IV was significant (P < 0.05). No significant difference in expression levels of EGF and VEGF was found among the 6 types (P > 0.05).

CONCLUSIONType I and II were the dominant TCM syndrome types in CAG patients; the high expression of PCNA might be a diagnostic evidence for Gan-Wei disharmony syndrome.

Adult ; Aged ; Aged, 80 and over ; Diagnosis, Differential ; Endothelial Growth Factors ; biosynthesis ; Female ; Gastric Mucosa ; metabolism ; pathology ; Gastritis, Atrophic ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Syndrome ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo study the effects of curcumin on the expression of the vascular endothelial growth factor (VEGF) and androgen-independent prostate cancer cell line PC-3, and to explore its anticarcinogenic mechanism.

METHODSPC-3 cells were treated with curcumin at the concentration of 0, 6.25, 12.5, 25 and 50 micromol/L respectively. Then the cell activity was assayed by dyed rate of Typan blue and MTT at 12, 24, 36, 48, 72 and 96 hours, the cell cycle and morphological changes observed by flow cytometry (FCM) and electronic microscopy at 24 hours, the VEGF mRNA expression measured by semi-quantitative RT-PCR, and the secreting protein levels of VEGF in the supernatants determined by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe growth of PC-3 cells was suppressed obviously by curcumin in a dose- and time-dependent manner in vitro. There were significant differences in inhibition rate among different concentration and time groups (P < 0.01). Furthermore, curcumin arrested the cell cycle of PC-3 cells in the G2/M phase in a dose-dependent manner (P < 0.01). The percentages of apoptotic cells were significantly higher in different concentration groups than in the controls (P < 0.01). Apoptosis-associated morphological changes were observed in PC-3 cells at 24 hours, and a marked decline in the expression of VEGF was noted after the exposure to different concentrations of curcumin within 24 hours.

CONCLUSIONCurcumin can suppress the growth of PC-3 cells, promote their apoptosis and arrest their cell cycle in the G2/M phase, and reduce the expression of VEGF mRNA and proteins, which may sever to explain its inhibitory effect on tumor and angiogenesis.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Curcumin ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Male ; Microscopy, Electron, Transmission ; Prostatic Neoplasms ; genetics ; pathology ; ultrastructure ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

In order to study the relation of antitumour mechanisms of triptolide with neovascularization, the effect of triptolide and tumour necrosis factor (TNF)-alpha on the expression of vascular endothelial growth factor (VEGF) in Raji cell lines and their effect on angiogenesis in human umbilical vein endothelial cells (HUVECs)-derived cell line ECV304 were investigated. The inhibitory rate of cells treated by triptolide detected by MTT; the ELISA was employed to study the VEGF content secreted by Raji cell lines; angiogenesis was tested by network formation of endothelial cells on Matrigel, and the mRNA level of VEGF was measured by RT-PCR. The results showed that treatment of Raji cells with triptolide resulted in significantly enhanced antiproliferative effects in dose- and time-dependent manner. The content of VEGF secreted by Raji cells was increased by TNF-alpha and was suppressed by triptolide (P < 0.01). The mRNA expressions of VEGF(165) and VEGF(121) (containing 165 and 121 amino acid residues, respectively) could be detected in all fractions. TNF-alpha augmented the expression of VEGF(165) and VEGF(121) mRNA when triptolide reduced the expression (P < 0.01). No network and cord were formed in control and triptolide group. There was tube formation on matrigel in the supernatants of Raji culture group and the supernatants groups treated by VEGF and TNF-alpha in Raji cell. It is concluded that the expressions of VEGF in Raji cells are increased by TNF-alpha and suppressed by triptolide. VEGF and TNF-alpha induce angiogenesis and triptolide inhibits angiogenesis in ECV304 cells.
Angiogenesis Inhibitors ; pharmacology ; Antineoplastic Agents, Alkylating ; pharmacology ; Cell Line ; Diterpenes ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Epoxy Compounds ; pharmacology ; Humans ; Lymphoma, B-Cell ; pathology ; Neovascularization, Physiologic ; drug effects ; Phenanthrenes ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; pharmacology ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of losartan (an angiotensin II type I receptor antagonist) on endothelial cells exposed to high glucose in vitro and related mechanism.

METHODSVascular endothelial cells of human umbilical vein were cultured in media with high glucose levels. The activities of SOD and CAT, the level of MDA were measured by spectrophotometry in the conditioned media of endothelial cells, the VEGF mRNA expression was performed using semi-quantitative reverse transcription PCR (RT-PCR) in the cell lysates, and the protein expression of VEGF was examined by enzyme-linked immunosorbent assay (ELISA) in the supernatants of cultured cells.

RESULTWhen endothelial cells were cultured in high glucose, the activities of SOD and CAT were significantly decreased, but the level of MDA was markedly increased. However, the high glucose-induced effects were inhibited by losartan. The application of high glucose upregulated the mRNA and protein expression of VEGF in endothelial cells, which was also attenuated by losartan.

CONCLUSIONHigh glucose disrupts the oxidative equilibrium and increases the expression of VEGF in endothelial cells, which can be inhibited by losartan.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Glucose ; pharmacology ; Humans ; Losartan ; pharmacology ; Peroxidase ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Superoxide Dismutase ; metabolism ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

OBJECTIVETo investigate the anti-angiogenic effect of amphiregulin (AR) antisense RNA expression in breast cancer.

METHODSHuman AR cDNA antisense plasmid was transfected into NS2T2A1 cells (a human breast cancer cell line). Two selected clones expressed AR antisense RNA (AR AS1 and AR AS3 cell lines) in which AR protein expression was reduced. Control cell line NS2T2A1 V was obtained by empty vector transfection. These cells were injected subcutaneously into nude mice. The effects of conditioned media on proliferation of human microvascular endothelial cells (HMEC) were evaluated and VEGF secreted by the cells was measured by ELISA method. In tumor tissues, VEGF expression levels were measured by quantitative RT-PCR, and CD31-immunostaining was used for intra-tumoral vascular quantification.

RESULTSThe proliferation index of HMEC cells grown in conditioned media with AR AS1 and AR AS3 was significantly reduced in comparison with that of control cells, accompanied by a decreased VEGF secretion. In tumors derived from AR AS1 and AR AS3 cells, intra-tumoral vascularization was reduced to about 50% of that derived from control cell line, accompanied with a decrease of VEGF expression.

CONCLUSIONAmphiregulin antisense RNA expression inhibits efficiently the angiogenesis in breast cancer, suggesting this growth factor could represent a novel therapeutic target in breast cancer.

Adenocarcinoma ; blood supply ; pathology ; Amphiregulin ; Angiogenesis Inhibitors ; biosynthesis ; genetics ; Animals ; Breast Neoplasms ; blood supply ; pathology ; EGF Family of Proteins ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Mice ; Mice, Nude ; Neovascularization, Pathologic ; RNA, Antisense ; biosynthesis ; genetics ; Vascular Endothelial Growth Factors ; antagonists & inhibitors

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.
Animals ; Body Weight ; Cell Survival ; Diabetes Mellitus, Experimental/*complications/metabolism ; Disease Models, Animal ; Glucose/pharmacology ; Glucose Tolerance Test ; Hyperglycemia/complications/*metabolism/*therapy ; Insulin/secretion ; Islets of Langerhans/blood supply/cytology/secretion ; *Islets of Langerhans Transplantation ; Liposomes/*administration & dosage/chemistry ; Male ; Mice ; Mice, Inbred BALB C ; Neovascularization, Physiologic ; RNA, Messenger/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Streptozocin ; Transfection ; Vascular Endothelial Growth Factors/biosynthesis/*genetics/*metabolism/secretion

OBJECTIVETo investigate the expression and pattern of vascular endothelial growth factor (VEGF) and its fetal liver kinase-1 (Flk-1) receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats.

METHODSForty-five adult male Wistar rats were divided randomly into a control group (n=5) and an experimental group (n=40). The bilateral sciatic nerves of the rats in the experimental group underwent neurotomy and the L4-L6 spinal cord and the corresponding dorsal root ganglia were harvested respectively at 8 hours, and 1, 3, 5, 7, 10, 14 and 21 days (8 subgroups with 5 rats each) after operation. The rats in the control group only underwent an exposure of sciatic nerve without neurotomy. Immunohistochemistry and image analysis were used to study the expression of VEGF and its Flk-1 receptor.

RESULTSBoth VEGF and Flk-1 receptor expressed in the normal rat spinal cord and dorsal root ganglia. In response to neurotomy, their expression reached a higher level and persisted for a short time then declined to the normal level rapidly. Besides, positive staining of Flk-1 was observed in both glial cells and nerve fibers, which located in the white matter of the spinal cord.

CONCLUSIONSVEGF can promote the regeneration of peripheral nerves from the angle of central neurons, which establishes the experimental and theoretical foundation for VEGF treating peripheral nerve injuries.

Analysis of Variance ; Animals ; Ganglia, Spinal ; metabolism ; Immunoenzyme Techniques ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Sciatic Nerve ; metabolism ; surgery ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; Vascular Endothelial Growth Factors ; biosynthesis

To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random. In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Bone Marrow Transplantation ; Cell Hypoxia ; Cells, Cultured ; Coronary Circulation ; Myocardial Infarction ; metabolism ; physiopathology ; surgery ; Neovascularization, Physiologic ; Random Allocation ; Rats ; Rats, Wistar ; Stromal Cells ; cytology ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

Vascular endothelial growth factor (VEGF) is a multi-functional cytokine involved in inflammation, repair and angiogenesis in asthmatic airway. This study aimed to evaluate the role of VEGF in immediate bronchoconstriction induced by TDI inhalation, and in chronic TDI-asthma patients. 11 newly diagnosed TDI-asthma patients (group I), 12 chronic TDI-asthma patients with persistent asthma symptoms followed for >4 yr and 15 unexposed healthy controls were enrolled. In group I, induced sputum and serum were collected before and 7 hr after placebo- and TDI-bronchoprovocation test (BPT). In group II, induced sputum and serum were collected every 2 yr. VEGF levels were measured by ELISA. There were no significant differences in sputum and serum VEGF levels between patients and controls. Before and after placebo and TDI-BPT, no significant changes were noted in sputum and serum VEGF levels of group I. In group II patients, sputum VEGF showed variable changes at 1-yr, then decreased significantly at 2-yr (p<0.05), while serum VEGF showed variable changes at 2-yr, which decreased significantly at 4-yr (p<0.05). These results suggest that VEGF may play a minor role in immediate bronchoconstriction after TDI-BPT. In chronic TDI-asthma, VEGF may be involved to 2 yr after the diagnosis and the contribution may decrease after then.
Adult ; Asthma/*chemically induced/*metabolism ; Bronchi/pathology ; Enzyme-Linked Immunosorbent Assay ; Exercise ; Human ; Methacholine Chloride/pharmacology ; Middle Aged ; Placebos ; Sputum/*metabolism ; Support, Non-U.S. Gov't ; Time Factors ; Toluene 2,4-Diisocyanate/*pharmacology ; Vascular Endothelial Growth Factor A/*biosynthesis/metabolism