Objective To introduce the studies on gene therapy for peripheral arterial disease(PAD) using plasmid DNA encoding human hepatocyte growth factor(HGF) gene. Methods Recent articles including preclinical and clinical studies were reviewed. Results Intramuscular injection of human HGF plasmid DNA into rat, rabbit, dog and diabetic hindlimb ischemic models, resulted in a significant increase in capillary density, blood flow and blood pressure. but no influence on tumor growth in mice. A clinical trial wasperformed in ischemic limbs of 6 critical limb ischemic patients, the result showed that no side effect caused by gene transfer was detected in all 6 patients.The pain scale and long diameter of ischemic ulcers were reduced. Conclusion Intramuscular injection of naked HGF plasmid DNA could be a safe and potential treatment for PAD.
Objective To evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors. Methods Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multipl icity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the prol iferation effect of HGF expression supernatant on BMSCs. Results Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% ± 0.04%, 40.72% ± 0.81%, 61.72% ± 1.04%, 85.33% ± 0.83%, and 17.91% ± 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the prol iferation of BMSCs. The prol iferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg). Conclusion BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the prol iferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.
Objective To investigate the possible association between serum level of hepatocyte growth factor( HGF) and obstructive sleep apnea hypopnea syndrome( OSAHS) with hypertension.Methods 58 cases of OSAHS without hypertension, 61 cases of OSAHS with hypertension, and 50 normal controls were enrolled. Serum level of HGF was measured by enzyme-linked immunosorbent assay( ELISA) , and the relationships between the serum HGF level and blood pressure( BP) , apnea hypopnea index( AHI) , lowest SaO2 ( LSaO2 ) were analyzed by linear correlation analysis. Results The serum HGF level ( pg/mL) was 761. 46 ±60. 18, 970. 87 ±60. 94, and 487. 34 ±45. 52 in the OSAHS patients without hypertention, OSAHS patients with hypertention, and normal subjects, respectively. Which was significantly higher in the OSAHSpatients than the normal subjects, and highest in the OSAHS patients with hypertension( P lt; 0. 05) . The serum HGF level was positively related to AHI( r = 0. 452, P lt;0. 05) and negatively related to LSaO2 ( r =- 0. 328, P lt;0. 05) in the OSAHS patients without hypertention, positively related to AHI, SBP, DBP( r =0. 670, P lt;0. 01; r =0. 535, P lt;0. 05; r =0. 424, P lt;0. 05) and negatively related to LSaO2 ( r = - 0. 572,P lt;0. 01) in the OSAHS patients with hypertension. Conclusions SerumHGF level increases significantly in patients with OSAHS especialy in OSAHS patients with hypertension, and positively correlates with the severity of OSAHS and hypertension.
ObjectiveTo study the expression of c-Met in colorectal carcinoma cells and the effect of hepatocyte growth factor (HGF) on proliferation and invasion of colon carcinoma cells SW480.
MethodsReal-time PCR and Western blot methods were respectively used to detect the expressions of c-Met mRNA and protein in the different colorectal carcinoma cells in order to screen the high c-Met expression cells. The SW480 cells were incubated with different concentrations (0, 20, 40, and 70 ng/mL) HGF. MTT assay and Transwell test were used to evaluate the effects of proliferation and invasion in the SW480 cells.
Results①The c-Met was expressed in each colorectal carcinomar cells, especially highly expressed in the colon carcinoma cells SW480 in vitro.②MTT assay showed that the HGF could promote the proliferation of SW480 cells in a dose-dependent manner with some extent.③Transwell test showed that the HGF could increase the invasion of SW480 cells.
ConclusionThe c-Met is highly expressed in colorectal carcinoma cells and HGF could promote proliferation and increase invasion of colorectal carcinoma cells in vitro.
Objective To investigate the effect of combined delivery of hepatocyte growth factor (HGF) and insulinlike growth factor-1 (IGF-1) on the development of bone mesenchymal stem cells (BMSCs) differentiation by expression of GATA-4，and to supply some evidence for clinical BMSCs transplantation therapy. Methods BMSCs were isolated from the femurs and tibias of the randomly assigned rabbits and cocultured with myocytes in a ratio of 1∶1. Myocytes were obtained from neonatal rabbits ventricles. 150 ng/ml HGF and 200 ng/ml IGF-1 were added into 4 culture bottles of 8 bottles and the other 4 bottles were not. After BMSCs were cocultured with myocytes for 1 day, 3 days, 1 week, and till 6 weeks， differentiated BMSCs were targeted and microdissected with a laser capture microdissection system, and then ribonucleic acid (RNA) was extracted and isolated. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcriptionpolymerase chain reaction (RT-PCR). And expression of GATA-4 in BMSCs was detected by semiquantitative RT-PCR. Results Before coculturing, the BMSCs were negative for α-actinin and exhibited a nucleus with many nucleoli. After coculture with myocytes, some BMSCs became αactininpositive and showed a cardiomyocytelike ultrastructure, including sarcomeres, endoplasmic reticulum, and mitochondria. BMSCs cocultured with myocytes expressed cardiac transcription factor GATA-4. IGF-1 and HGF delivery can significantly increased expression of GATA-4 for the differentiated BMSCs as compared with cells of no delivery of HGF and IGF-1. The expression level of GATA-4 in captured BMSCs began to increase at the 1st day, reach the peak at the 2nd week and kept high expression level after the 2nd week. Conclusion BMSCs can transdifferentiate into cells with a cardiac phenotype when cocultured with myocytes. Differentiated myocytes express cardiac transcription factors GATA-4. Administration of HGF and IGF-1 promoted the development of BMSCs transdifferentiate into cardiac phenotype, which is associated with the increase in expression level of GATA-4.
Objective To study hepatocyte growth factor (HGF) and its receptor (c-met) expressions in human colorectal cancer and non-neoplasm colorectal mucosa, and the relationship with tumor angiogenesis. Methods Tissue microarrays (TMAs) were made up of 80 cases of colorectal cancer and 80 cases of nonneoplasm colorectal mucosa. The expressions of HGF and c-met were detected by immunohistochemistry (SP). CD105 was used as a marker to account microvessel density (MVD) in tumor tissue. Results HGF was over expressed in 48 cases and c-met was over expressed in 63 cases of colorectal cancer tissue, and the correlation between HGF and c-met positive expression was significant (r=0.231, Plt;0.05). The high expression rate of HGF and cmet in colorectal cancer were significantly higher than that in non-neoplasm colorectal mucosa (χ2=35.387, Plt;0.05; χ2=59.854, Plt;0.05) of colorectal cancer. The overexpression of HGF was correlated with lymph node metastasis (χ2=4.743, Plt;0.05) and TNM staging (χ2=5.576, Plt;0.05). The overexpression of c-met was correlated with differentiation (χ2=15.767, Plt;0.05) and lymph node metastasis (χ2=5.765, Plt;0.05) of colorectal cancer. MVD was different between overexpression and lowexpression colorectal cancer tissues of HGF and cmet (t=2.150, Plt;0.05; t=2.052, Plt;0.05). There was statistical correlation between HGF and cmet overexpression (r=0.259, Plt;0.05). The overexpressions of HGF and cmet were correlated with lymph metastasis in moderate differentiation cancer (χ2=13.154, Plt;0.05; χ2=5.371, Plt;0.05). Conclusions The overexpressions of HGF and c-met in colorectal cancer may be related with tumor angiogenesis. Detecting the expressions of HGF and c-met is valuable to estimate the biological character of colorectal cancer.
Objective To study the effects of hepatocyte growth factor (HGF) and receptor c-met on the development of primary breast carcinoma, and the relationship between it and prognosis. Methods The study of HGF and c-met related to breast carcinoma was reviewed by history document and experimental study in recent years. Results HGF is a growth factor which has mitogenic, migrating, invasive and angiogenic activities in breast carcinoma cells. The carcinogenic mechanism of breast carcinoma was more clear with the discovery of the relationship between HGF and its receptor c-met. Conclusion The HGF/c-met plays an important role in the generation and progress of breast carcinoma. Studying the effects of HGF/cmet on breast carcinoma is significant in guiding clinical treatment.
Objective To evaluate the effect of hepatocyte growth factor(HGF) on intestinal permeability and bacterial translocation after small bowel transplantation in rats. Methods Twenty Wistar rats were as recptors and twenty SD rats as donors. After heterotopic intestinal grafting, cyclosporine A was administered at 6mg/kg·day intramuscularly for inhibiting rejection. The SD rats were divided into 2 groups(n=10). HGF was administered at 150 μg/kg·day (HGF group) and normal saline was administered at 150 μg/kg·day (controlgroup). Intestinal permeability and bacterial translocation to the mesenteric lymph nodes and portal vein were assessed at the 8th postoperative day. Results The lactulose and lactulose/ mannitol of control group (0.0931%±0.008 5% and 0.132± 0.021) were higher than those of normal reference value (0.015 0%±0.002 0% and 0.020±0.005)（Plt;0.05）. The lactulose and lactulose/ mannitol of HGF group (0.039 6%±0.009 0% and 0.056±0.013) were also higher than those of normal reference value（Plt;0.05）.The bacterial culture positive proportion of lymphaden in HGF group and control group were 10% and 60%, showing statistically significant difference（Plt;0.05）. The bacterial culture positive proportion of portal vein in HGF group and control group were 10% and 20% respectively（P＞0.05）. Conclusion HGF can decrease intestinal permeability and bacterial translocation from the lumen of the graft to the mesenteric lymph nodes,thus improve gut barrier function, may be of help to reduce the incidence of septic complications after intestinal grafting.
Objective
To investigate the effect of HGF on proliferation and migration in cultured human RPE cells.
Methods
Human RPE cells cultured in serum-free medium were treated with HGF(1，2，10，50，100 mu;g/L), and MT T assay was used to detect the growth of the cells; an in vitro wound healing model was used to count the number of cells that had entered the denudate area in RPE mig ration treated with HGF (1，2，10，50，100 mu;g/L) after 20 h.
Results
HGF(10,50,100 mu;g/L) increased proliferation rates of cultur ed human RPE (18.2 % to 34.8 %), and at a concentration of 50 mu;g/L on day 3 HGF induced the maximal increase of proliferation (Plt;0.01）; HGF showed effects on migration of 9.3 %, 113.0 %, 91.7 % and 50.3 % at the concentrations of 2,10,50, 100 mu;g/L, respectively. HGF stimulated t he best migratory response in RPE cells at a concentration of 10 mu;g/L (113 %).
Conclusion
HGF can induce the proliferation and obvious migration of RPE cells, consequently HGF was a mitogen and potent migratory factor for human cultured RPE cells.
(Chin J Ocul Fundus Dis, 2001,17:307-310)
Abstract： Objective To investigate the effects of hepatocyte growth factor（HGF）gene transfected bone marrow mesenchymal stem cells （MSCs）transplantation in pigs with chronic ischemic heart disease. Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture, purified, and determined by cellsurface antigens（CD34, CD44, CD71, Ⅷ factor and desmin）. MSCs were transfected by adenovirus expressing hepatocyte growth factor（AdHGF）, and the influence of HGF on the biological characteristics of MSCs was tested. The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via leftthoracotomy. A total of 40 pigs were randomly divided into 5 groups （n=8） and were injected 5×106/ml MSCs＋ 4×109 pfu 200 μl AdHGF （MSCs＋ AdHGF group）, 4×109 pfu 200 μl AdHGF （AdHGF group）, 5×106/ml MSCs 200 μl（MSCs group），4×109 pfu 200 μl AdNull （AdNull group）and 1 ml saline（control group） into the ischemic myocardiumrespectively. Echocardiogram, digital subtraction angiography （DSA） of coronary artery, single photon emission computed tomography（SPECT） myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks. Results Positive CD44 and CD71 and negative CD34, Ⅷ factorand desmin were detected in MSCs by flow cytometer. HGF had a b influence on stimulating the proliferation and differentiation of MSCs. Echocardiogram examination showed that left ventricular end-diastolic volume（LVEDV），left ventricular ejection fraction（LVEF），fractional shortening（FS）of MSCs＋ AdHGF group were significantly increased after treatment （P＜ 0.05）. DSA detection showed that ischemic neovascularization of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group （P＜ 0.05）. SPECT showed that the left ventricular myocardium of MSCs＋ AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased （P＜ 0.05）. Vascular density of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium ［（39.4±1.2）/ HPF vs. （36.5±1.4）/ HPF and（34.5±1.7）/ HPF，P＜ 0.05］. Cardiomyocyte apoptosis rate of MSCs＋ AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain （P＜ 0.05）. Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium, inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease. The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.
