To investigate the effect of hepatocyte growth factor (HGF) on prol iferation of cultured human eccrine sweat gland epithel ial cells (hESGc) and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the prol iferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 103 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGcwere cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF incontrol group, and in KSFM without HGF and no hESGc in blank group. The cell prol iferation was observed in xperimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the prol iferation of hESGc (P lt; 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell prol iferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.074 3 at 4 days; showing significant differences when compared with control group (P lt; 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell prol iferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.231 0 ± 0.056 7 at 4 days; showing significant differences when compared with control group (P lt; 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation (P lt; 0.01). Conclusion HGF could induce the prol iferation of hESGc and activate the phosphorylation of ERK1/2 protein.
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 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 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 explore the effects of bone marrow mesenchymal stem cells (BMSCs) transfected with adenovirus hepatocyte growth factor (Ad-HGF) on wound repair in diabetic rats. Methods BMSCs from male Wistar rats were isolated by density gradient centrifugation, cultured, and transfected with Ad-HGF. The multi pl icity of infection was 100. Diabetic models were establ ished in 20 female Wistar rats by diets in high fat and sugar plus intraperitoneal injection ofstreptozotocin (30 mg/kg). Then 2 full-thickness skin wounds (approximately 1.5 cm in diameter) were made on the dorsum. The rats were randomly divided into 4 groups (n=5 rats). After wounding, the 0.3 mL suspensions of BMSCs (group A), Ad- HGF (group B), BMSCs transfected with Ad-HGF (group C), and PBS (group D) were injected directly into the derma of wounds. The transverse diameter and longitudinal diameter of wound were measured at 21 days after treatment. At 7 days and 28 days after treatment, HE staining was performed to evaluate wound heal ing. The contents of hydroxyprol ine and advanced glycosylation end products (AGEs) in the wounds were measured by enzyme l inked immunosorbent assay and fluorospectrophotometer, respectively, at 3, 7, 14, and 28 days after treatment. Results At 21 days after treatment, the wounds almost healed in group C, and the transverse diameter and longitudinal diameter were 0 and (0.110 ± 0.024) cm, respectively. But the wounds healed partially in groups A, B, and D, and the transverse diameter and longitudinal diameter were (0.470 ± 0.051) cm and (0.590 ± 0.041) cm, (0.390 ± 0.042) cm and (0.480 ± 0.032) cm, and (0.700 ± 0.068) cm and (0.820 ± 0.068) cm, respectively. There were significant differences in wound heal ing between group C and groups A, B, and D (P lt; 0.05). The wound heal ing time of group C [(20.5 ± 1.9) days] was significantly shorter (P lt; 0.05) than those of groups A, B, and D [(28.3 ± 1.9), (25.9 ± 2.3), and (36.6 ± 5.1) days]. At 7 days, the HE staining showed that evident epidermis transportation, collagen formation, and leukocytes infiltration were observed in group C. At 28 days, the HE staining showed that the epidermis in group C was significantly thinner and more regular than those in other groups, and the decreased collagen and many small vessels were observed in group C. The content of hydroxyprol ine in group C was higher than those in groups A, B, and D at 7 days and 14 days (P lt; 0.05). The contents of AGEs in group C was lower than those in groups A, B, and D at 14 days and 28 days (P lt; 0.05). Conclusion Transplantation of BMSCs transfected with Ad-HGF can accelerate the wounds repair in diabetic rats.
Abstract: Objective To explore the significance of peripheral serum hepatocyte growth factor (HGF) and transforming growth factor-β (TGF-β) in preoperative staging of patients with nonsmall cell lung cancer. Methods Fifty patients, including 30 males and 20 females, with complete clinical data and final pathological diagnosis of nonsmall cell lung cancer were treated in Beijing Chaoyang Hospital from September 2006 to November 2007. Their age ranged from 36 to 76 years old (62.4±10.0 years old). Among the patients, there were 26 patients of adenocarcinoma, 23 patients of squamous cell carcinoma and one patient of large cell carcinoma. Twenty other normal subjects were chosen to form normal control, including 11 males and 9 females, aged from 18 to 67 years old (43.8±14.2 years old). Peripheral serum HGF and TGF-β were measured with enzymelinked immunosorbent assay (ELISA), and the relationship between the level of HGF, TGF-β and preoperative staging was analyzed. Results The peripheral serum HGF and TGF-β level has no relation with patient’s age, sex, smoking history or histology type. The level of HGF in the T2 and T3 patients was significantly higher than that of normal control (373.90±234.00 pg/ml vs. 211.30±154.60 pg/ml, t=2.759, P=0008; 563.80±316.10 pg/ml vs. 211.30±154.60 pg/ml, t=4076, P=0.000). The level of TGF-β in the T-3 patients was significantly higher than that of normal control (3.34±2.80 ng/ml vs. 1.82±0.90 ng/ml, t=2.190, P=0.037). The level of TGF-β in the N1-2 patients was significantly higher than that of the N0 patients (2.60±2.00 ng/ml vs. 1.53±0.74 ng/ml, t=-2.387, P=0.021). TGF-β level (5.97±2.65 ng/ml) in patients with distant metastasis (stage Ⅳ) was significantly higher than that of patients in other stages. Conclusion The HGF and TGF-β level is related to the staging of lung cancer. Such examinations combined before operation may present a reference value for preoperative staging and providing the best treatment plan for the patients.
Objective To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor (hHGF) gene-modified bone marrow mesenchymal stem cells (BMSCs) on the immunological rejection after allograft l iver transplantation in rats, and to reveal the mechanism of immune tolerance. Methods Eight male Sprague Dawley (SD)rats of clean grade (aged 3 to 4 weeks, weighing 75-85 g) were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade (weighing 200-250 g) were used as donors; and 64 adult male Wistar rats of clean grade (weighing 230-280 g) were used as receptors. After establ ishing a stable model of rat allogeneic l iver transplantation, 1 mL sal ine, 2 ×106/mL of BMSCs 1 mL, 2 × 106/mL of BMSCs/green fluorescent protein 1 mL, and 2 × 106/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the l iver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 (IL-2), IL-4, IL-10, and interferon γ (IFN-γ) by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of prol iferating cell nuclear antigen (PCNA) by immunohistochemical method. Results The survival time of group D was significantly higher than that of groups A, B, and C (P lt; 0.01); the survival time of groups B and C was significantly higher than that of group A (P lt; 0.01), but there was no significant difference between group B and group C (P gt; 0.05). RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to (6.2 ± 1.0) ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved l iver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-γ decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 (P gt; 0.05), there were significant differences in the other indexes between group D and groups B, C (P lt; 0.05). Conclusion BMSCs/hHGF implanting to rat l iver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Th1/Th2 shift, reduced apoptosis, promoted l iver regeneration.
Objective To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect. Methods BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfectedwith Ad-HGF. The MI models were establ ished in 44 SD male rats (weighing 200-250 g) by l igating the left coronary artery. At 4 weeks after l igation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 × 107/ mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n=8). The 0.1 mL normal sal ine was injected into the infarct zone in control group; 0.1 mL normal sal ine was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining. Results The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systol ic pressure (LVSP), left ventricular end-diastol ic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P gt; 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P lt; 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P lt; 0.05). Immunofluorescence staining showed that the vascular endothel ial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapygroup than in other 3 groups (P lt; 0.05). Conclusion The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
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.