OBJECTIVETo investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose.

METHODSThe third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (10(-7)-10(-3) mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-β1), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-β1 and HGF mRNA expression by reverse transcription polymerase chain reaction.

RESULTSCpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P<0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-β1 were reduced markedly (P<0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10(-5) mmol/L).

CONCLUSIONThe anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type IV ; biosynthesis ; secretion ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Glucose ; toxicity ; Hepatocyte Growth Factor ; secretion ; Matrix Metalloproteinase 9 ; metabolism ; Mesangial Cells ; cytology ; drug effects ; enzymology ; metabolism ; Polymerase Chain Reaction ; Proteolysis ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transforming Growth Factor beta1 ; secretion

Hepatocyte growth factor (HGF) is a potent angiogenic factor that can stimulate the production of blood vessels in ischemic tissue. We investigated whether gene therapy using HGF-expressing adenovirus could enhance skin flap survival. Sprague-Dawley rats were randomly divided into three groups. Rats were subdermally injected with HGF-expressing adenovirus (HGF virus group), recombinant HGF (rhHGF group), or phosphate buffered saline (PBS group) 2 days before and immediately after 3 x 9 cm caudal flap elevation. The survival area of the skin flap, the ratio of blood flow, CD31-positive vessels and, VEGF expression were examined. Skin flap viability was significantly increased in the HGF virus group compared to the rhHGF and PBS groups (71.4% +/- 5.9%, 63.8%+/- 6.4%, and 39.2% +/- 13.0%, respectively) (P = 0.025). Furthermore, the blood flow ratio was significantly increased in the HGF virus group. In the HGF virus group, the number of CD31-positive vessels and vascular endothelial growth factor (VEGF) expression were significantly increased. Gene therapy using HGF-expressing adenovirus increase VEGF expression, the number of viable capillaries, and blood flow to the flap, thereby improving skin flap survival.
Adenoviridae/genetics ; Animals ; Genetic Therapy/*methods ; Graft Survival/genetics ; Hepatocyte Growth Factor/biosynthesis/*genetics ; Male ; Models, Animal ; Neovascularization, Physiologic/*genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reconstructive Surgical Procedures ; Skin Transplantation/*methods ; Surgical Flaps/*surgery

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

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

OBJECTIVETo investigate the biological effect of hepatocyte growth factor (HGF) on HGF gene-transfected Raji cells.

METHODSTotal RNA was extracted from human hepatic tissue, HGF gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF. The recombinant vector was transfected to Raji cells, and the stably transfected cells were selected by homomycin B in serial passages, and confirmed by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry. The biological features of transfected Raji cells were evaluated by semisolid culture.

RESULTSRT-PCR results showed that Raji cells were transfected successfully with recombinant eukaryotic expression vector pVITRO2-mcs-HGF. HGF mRNA and protein were expressed successfully in Raji cells. Expression of HGF gene enhanced proliferation, metastasis and invasion of Raji cells.

CONCLUSIONHGF gene has been cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF successfully. Transfected HGF may change the biological features of Raji cells.

Cell Line, Tumor ; Cloning, Molecular ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Lymphoma, B-Cell ; genetics ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

In order to trace the expression of exogenous hepatocyte growth factor (HGF) in eukaryotic cells exactly, a recombinant plasmid that expresses the fusion protein of HGF and red fluorescent protein (RFP) was constructed. The gene encoding HGF without stop code was amplified from pMD19-T-HGF by PCR technique and then cloned in pDsRed-express-N1. The recombinant plasmid (pDsRed-HGF) was identified by restriction endonuclease enzyme analysis and DNA sequence analysis. pDsRed-HGF was transfected into 293T cells. The expression of HGF mRNA, HGF and red fluorescent protein was detected. The results showed that the target gene sequence in pDsRed-HGF was completely in conformity with HGF cDNA in GenBank, and it was expressed highly in 293T cells. In this study, pDsRed-HGF was successfully constructed and expressed in eukaryotic cells.
Cell Line ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Luminescent Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo study the effect and mechanism of Kangxianling (KXL, a TCM herbal compound) on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO).

METHODSEighteen male SD rats were randomly divided into 3 groups, 6 in each group, the sham operated group, the model group, and the KXL group. Renal interstitial fibrosis model was established in rats by UUO. After rats were raised for additional 14 days, their body weight, serum levels of creatinine (SCr) and blood urea nitrogen (BUN) were analyzed. Then rats were sacrificed, their renal pathology examined by HE staining and PASM staining; expressions of transforming growth factor-beta1 (TGF-beta1), hepatocyte growth factor (HGF) mRNA, and a-smooth muscle actin (alpha-SMA), TGF-beta1 receptor I (TbetaR I), TGF-beta1 receptor II (TbetaR II) and hepatocyte growth factor receptor (C-Met) protein in kidney tissue were determined by RT-PCR and Western blotting respectively.

RESULTSSCr and BUN in the model group were significantly higher than those in the sham operated group (P <0.05). Expressions of TGF-beta1 mRNA and a-SMA, TbetaR I , TbetaR II and C-Met protein in kidney tissue in the model group significantly up-regulated and mRNA expression of HGF significantly down-regulated, and obvious hyperplasia of the base member of glomeruli was seen. After intervention with KXL, BUN content significantly lowered, alpha-SMA, TbetaR I and TbetaR II protein expression decreased and HGF mRNA expression up-regulated significantly in the treated group, with slight pathological changes only shown as mild hyperplasia of the base member of glomeruli and renal tubules.

CONCLUSIONKXL could inhibit the protein expressions of a-SMA, TbetaR I , TbetaR II and increase the mRNA expression of HGF, which is a protective factor against renal fibrosis. Therefore, it is effective in alleviating the renal interstitial fibrosis and improving the renal function in UUO rats.

Animals ; Blotting, Western ; Drugs, Chinese Herbal ; therapeutic use ; Fibrosis ; prevention & control ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Kidney ; drug effects ; metabolism ; pathology ; Male ; Nephritis, Interstitial ; etiology ; pathology ; prevention & control ; Nephrosclerosis ; pathology ; prevention & control ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Ureteral Obstruction ; complications

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

OBJECTIVETo investigate the protective effect of hepatocyte growth factor (HGF) on protein synthesis in rat cardiomyocytes exposed to gamma-ray irradiation.

METHODSPrimary cultured cardiomyocytes were irradiated with single-dose (20 Gy) gamma ray in the absence or presence of HGF (40 ng/ml) added in the cell culture 3 h before the exposure. Forty-eight hours after irradiation, the total cellular protein was measured and cell cycle analyzed by flow cytometry. The cardiomyoctes were also infected with AdGFP 48 h after irradiation and the fluorescence intensity of the green fluorescence protein (GFP) in the cells determined by flow cytometry 48 h after infection.

RESULTSThe protein synthesis was decreased significantly in the irradiated cardiomyocytes as compared with the control group (P<0.01), but was remedied significantly by incubation of the cells with HGF before the exposure (P<0.05). Flow cytometry revealed much lower mean fluorescence intensity (MFI) of GFP in irradiated cardiomycytes than in cells without the exposure (P<0.01); The MFI was higher in HGF-treated cardiomyocytes than in cells without HGF treatment following the exposure (P<0.01).

CONCLUSIONGamma ray irradiation inhibits protein synthesis in cardiomyocytes, and HGF may attenuate this effect of gamma ray exposure for cardiomyocyte protection.

Animals ; Animals, Newborn ; Cell Cycle ; drug effects ; radiation effects ; Cells, Cultured ; Flow Cytometry ; Gamma Rays ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; Microscopy, Fluorescence ; Myocytes, Cardiac ; cytology ; metabolism ; Protein Biosynthesis ; drug effects ; radiation effects ; Rats ; Rats, Wistar

PURPOSE: Hepatocyte growth factor (HGF) and its receptor (HGFR/c-Met) regulate motility, mitogenesis, and morphogenesis in a cell type-dependent fashion. We report the role of HGF and c-Met on stress-induced ARPE-19 human retinal pigment epithelial (RPE) cells in this study. METHODS: The cells were cultured either with or without serum. Southern and Western blot analyses were done to determine the expression patterns of HGF/c-Met in serum-starved ARPE-19 cells. The cell proliferation pattern in serum-starved condition was analyzed using MTS assay. Inhibition level of cell proliferation was analyzed using a neutralizing monoclonal antibody against c-Met (2 microgram/ml). RESULTS: Abnormal cell proliferation and scattering of ARPE-19 cells was observed under serum starvation. HGF/c-Met were expressed in serum-starved ARPE-19 cells. ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment. Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%). Serum starvation appears to induce epithelial-mesenchymal transition of ARPE-19 cells, resulting in scatter, and the expression of alpha-smooth muscle actin (alpha-SMA), a marker for fibrosis. CONCLUSIONS: In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.
Blotting, Southern ; Blotting, Western ; Cell Movement/physiology ; Cell Proliferation ; Cells, Cultured ; Culture Media, Serum-Free ; *Gene Expression ; Hepatocyte Growth Factor/biosynthesis/*genetics ; Humans ; Mitosis/physiology ; Pigment Epithelium of Eye/cytology/*metabolism ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-met/biosynthesis/*genetics ; RNA/*genetics