Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

AIMTo construct a plasmid carrying hepatocyte growth factor gene and investigate its effects in vitro.

METHODSA complementary DNA (cDNA) clone for human HGF was isolated from human placental cDNA, then subcloned into pUDK vector, which was constructed by ourselves, to form the pUDKH plasmid. The transfection efficiency and the expression level of HGF and VEGF were evaluated by transfecting pUDK or pUDKH into primary rat skeletal muscle cells. The biological effects of HGF-expressing product at different doses on endothelial cells were investigated in vitro, and assessed by MTT.

RESULTSThe primary rat skeletal muscle cells could be transfected efficiently with pUDKH (0.057%), and secreted HGF(16 -18 ng/4 x 10(5) cells) and VEGF proteins. The expressing product could significantly stimulate proliferation of human umbilical vein endothelial cells, in a dose-dependent manner (P < 0.05).

CONCLUSIONpUDKH has the potential application in vivo to treat ischemic diseases.

Animals ; Cells, Cultured ; DNA, Complementary ; genetics ; Genetic Vectors ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Plasmids ; Rats ; Rats, Wistar ; Transfection

OBJECTIVETo obtain high expression of human hepatocyte growth factor (HGF) in passaged rabbit bone marrow-derived mesenchymal stem cells (BMSCs) via recombinant adenovirus vector mediated HGF gene transfection, and explore the feasibility of this strategy for local treatment of avascular necrosis of the femoral head (ANFH).

METHODSHGF gene was subcloned into the adenovirus shuttle plasmid pDC316, and the products were co-transfected into HEK293 cells with the helper plasmid pBHGlox deltaE1,3Cre. The recombinant adenovirus Ad-HGF was generated by homologous recombination of the 2 plasmids in HEK293 cells. After PCR identification, Ad-HGF was amplified and purified and its titer measured by TCID50 assay before transfected into the second passage of rabbit BMSCs. The transcription and expression of HGF gene in the transfected BMSCs was detected by RT-PCR, in situ hybridization, and immunological histochemistry.

RESULTSAd-HGF was successfully constructed with a titer of 2.6x10(10) TCID50/ml. The expressions of HGF mRNA and protein were confirmed in the transfected BMSCs.

CONCLUSIONAd-HGF transfection allows efficient expression of HGF protein in rabbit BMSCs, and this success may facilitate further study of local treatment of ANFH using HGF-expressing BMSCs.

Adenoviridae ; genetics ; Animals ; Cell Line ; Genetic Vectors ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Rabbits ; Transfection

BACKGROUND: Hemoglobin concentration reportedly is positively associated with muscle strength, for example, handgrip strength. However, hemoglobin cannot repair muscle directly, but is beneficial only in a supportive role. Since hepatocyte growth factor (HGF) regulates muscle satellite cell production and differentiation, which is stimulated by organ injury, the supportive effect of hemoglobin should thus be stronger for participants with high HGF than for those with low HGF. However, the association between hemoglobin concentration and handgrip strength in relation to HGF levels remains unknown. METHODS: We conducted a cross-sectional study of 255 Japanese elderly men aged 60-69 years who participated in annual health check-ups in 2014-2015. The study population was categorized on the basis of a median value of HGF of 300.6 pg/mL. RESULTS: Among present study population, 128 participants showed low HGF. For participants with low HGF, hemoglobin concentration showed no significant association with handgrip strength (standardized parameter estimate (β) = 0.03, p = 0.767), but for those with high HGF, hemoglobin concentration was significantly positively associated with handgrip strength (β = 0.23, p = 0.014). CONCLUSIONS A significant positive association between hemoglobin level and handgrip strength was established for elderly Japanese men aged 60-69 years with high HGF but not for participants with low HGF. Our finding indicates that HGF levels could determine the relationship of hemoglobin concentration with handgrip strength in elderly Japanese men aged 60-69 years. This result can be expected to serve as an effective tool for the clarification of the roles played by HGF and hemoglobin concentration in maintenance of muscle strength.
Aged ; Cross-Sectional Studies ; Hand Strength ; physiology ; Hemoglobins ; metabolism ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Japan ; Male ; Middle Aged

Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; Hepatocyte Growth Factor ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism

The present investigation was to construct lentiviral vector carrying the human gene NK4 and transfect the human bone mesenchymal stem cells (hBMSCs) and to determine the expression of NK4 gene in hBMSCs after transfection. The NK4 gene was obtained from HGF cDNA by polymerase chain reaction(PCR), and the pGC-FU-NK4 plasmid was constructed by double restriction enzyme digestion and gene recombinant. The titer of virus was tested by real-time quantitative PCR. After transfected by lentivirus, the green fluorescent protein (GFP) in hBMSCs was observed using fluorescence microscope, and the expression of NK4 in culture supernatant was detected by enzymelinked immunosorbent assay (ELISA). The sequence of the PCR product was consistent with the data of GeneBank by DNA sequencing. The virus titer was 2 X 10(8)TU/ml. Strong green fluorescence was observed in the cell membrane and cytoplasm of hBMSCs with fluorescent microscopy. The expression of NK4 in culture supernatant was increased with time extension. The hBMSCs can be transfected by NK4 gene expressing lentiviral vector safely and effectively, and the expressin and secretion of NK4 was persistent and stable.
Bone Marrow Cells ; metabolism ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells.

METHODSPlasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay.

RESULTSPlasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05).

CONCLUSIONOur findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.

Apoptosis ; Cell Cycle Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; HeLa Cells ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Plasmids ; Proto-Oncogene Proteins ; genetics ; metabolism ; Transfection

OBJECTIVE: To study the expressions of FSCN1 and HGF in nasal inverted papilloma (NIP) and explore their role in occurrence and development of this disease. METHOD: Immunohistochemical method was used to determine the expression of FSCN1 and HGF in 12 cases of chronic hypertrophic rhinitis, 40 cases of NIP and 14 cases of NIP with malignant transformation. RESULT: FSCN1 was expressed in 52.5% of NIP, 78.6% of NIP with malignant transformation and 8.3% of inferior turbinate of chronic hypertrophic rhinitis. Expression of FSCN1 was significantly higher in NIP and NIP with malignant transformation than in inferior turbinate (P<0.05). HGF was expressed in 85.7% of NIP with malignant transformation and 8.3% of inferior turbinate. Expression of HGF was significantly higher in NIP with malignant transformation than in inferior turbinate (P<0.05). HGF was expressed in 40.0% of NIP,which was higher than that of inferior turbinate. Expression of HGF was positively related to expression of FSCN1 in NIP and NIP with malignant transformation. CONCLUSION The abnormal expression of FSCN1 and HGF may be closely correlated with NIP and its malignant process. Analysis of FSCN1 and HGF expression in NIP may be useful in predicting malignant transformation.
Carrier Proteins ; genetics ; metabolism ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Nasal Mucosa ; metabolism ; pathology ; Nose Neoplasms ; genetics ; metabolism ; pathology ; Papilloma, Inverted ; genetics ; metabolism ; pathology ; Turbinates ; metabolism ; pathology

pUDK-HGF, the recombinant plasmid DNA encoding human hepatocyte growth factor (HGF), can treat ischaemic disease. A great quantity of pharmaceutical pUDK-HGF is needed. A pilot-scale production process of pUDK-HGF was established based on a new chromatographic media (plasmidselect), including fermentation, cell harvesting, alkaline lysis, ultrafiltration, RNA removing and buffer exchanging on Sephacryl S-1000, capturing supercoiled plasmid DNA with plasmidselect, and removing the salt with Sepharose 6BFF. The process does not use RNase enzyme and toxic solvents.
DNA, Recombinant ; biosynthesis ; DNA, Superhelical ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Pilot Projects ; Plasmids ; isolation & purification

BACKGROUNDHepatocyte growth factor (HGF) inhibits the development of pulmonary artery hypertension (PAH) by reducing pulmonary artery pressure and right ventricle (RV) hypertrophy. However, whether HGF can prevent RV remodeling via inhibiting apoptosis in RV cardiomyocytes and decreasing neurohormonal activation remains unknown.

METHODSThe PAH and subsequent RV remodeling in rats were induced by subcutaneous injection of monocrotaline (MCT). The PAH rats were transfected with adenovirus carrying HGF (Ad-HGF) via intratracheal instillation. Three weeks after transfection, the hemodynamics indexes were measured, serum levels for angiotonin II (ANG II) and brain natriuretic peptide (BNP) were determined by ELISA. Histological analysis was used to assess the RV hypertrophy and fibrosis. The cardiomyocyte apoptosis in RV was assayed by TUNEL staining. The mRNA expression of BNP, angiotensin-converting enzyme (ACE), Bax and Bcl-2 in RV was determined by reverse transcriptase polymerase chain reaction (RT-PCR), the protein expression of transforming growth factor (TGF)-β1 and tumor necrosis factor (TNF)-α in RV was determined by Western blotting.

RESULTSHGF treatment significantly decreased the mean PAH, RV systolic pressure, serum ANG II and BNP levels. HGF treatment also significantly decreased the RV hypertrophy, collagen deposition, and the number of apoptotic cardiomyocytes. Moreover, HGF treatmemt significantly decreased the expression of BNP, ACE, Bax, TGF-β1, and TNF-α, while it significantly increased the expression of Bcl-2.

CONCLUSIONSGene transfer of HGF decreases MCT-induced PAH and improves RV remodeling. This effect is mediated not only by improving the hemodynamics but also by decreasing neurohormonal activation and inhibiting cardiomyocytes apoptosis. HGF gene treatment may be an effective strategy for improving RV remodeling in MCT-induced PAH.

Animals ; Apoptosis ; genetics ; physiology ; Hepatocyte Growth Factor ; genetics ; physiology ; therapeutic use ; Humans ; Hypertension, Pulmonary ; metabolism ; therapy ; Male ; Rats ; Rats, Sprague-Dawley ; Ventricular Remodeling ; genetics ; physiology