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.
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.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
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)
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.
【Abstract】ObjectiveTo explore the effect of hepatocyte growth factor/scatter factor (HGF/SF) on apoptosis of colorectal cancer cells induced with curcumin. MethodsMTT assay was used to evaluate the cytotoxicity of curcumin to colorectal cancer cells. Flow cytometry was used to detect the antiapoptosis effect of HGF. ResultsFlow cytometry showed only 64 μg/ml curcumin could play the proliferationinhibiting role in Caco-2 cells leading to their apoptosis; at the same time, different concentrations of HGF could antagonize this inhibitory effect resulting in the decrease of apoptosis, but HGF worked without a concentration-dependent manner. The study on MAPK pathway showed that the protective effect of HGF on the apoptosis of Caco-2 cells was not influenced by inhibiting p42/p44 MAPK and p38 MAPK pathway. ConclusionHGF/SF antagonizes the apoptosis of Caco-2 cells induced with curcumin, but MAPK signaling pathway might not participate in this process.
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 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 construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multipl icities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 108 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
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.