Animals ; Estradiol ; blood ; pharmacology ; Hepatocyte Growth Factor ; metabolism ; Liver Cirrhosis ; metabolism ; pathology ; Male ; Rats ; Rats, Wistar

OBJECTIVETo explore the effect of tacrolimus on murine hair follicle cycle.

METHODHematoxylin-eosin dyeing and reverse transcription-polymerase chain raction techniques were used.

RESULTSFive days after depilation, the hair follicles in both the tacrolimus group and the minoxidil group was in anagen V, while that in the vaseline group was in anagen III. vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were detected in back skin in both the tacrolimus group and the minoxidil group, but not in the vaseline group.

CONCLUSIONTacrolimus can promote the growth of hair by stimulating the hair follicle to enter anagen V in mice, which may be explained by the effects of VEGF and HGF.

Animals ; Hair Follicle ; drug effects ; physiology ; Hepatocyte Growth Factor ; metabolism ; Mice ; Mice, Inbred C57BL ; Minoxidil ; pharmacology ; Skin ; drug effects ; metabolism ; Tacrolimus ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs).

METHODSADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry.

RESULTSThe secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01).

CONCLUSIONSInsulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.

Adipocytes ; cytology ; drug effects ; secretion ; Cells, Cultured ; Fibroblasts ; cytology ; Hepatocyte Growth Factor ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; drug effects ; secretion ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the inhibitory effect of hepatocyte growth factor (HGF) on the apoptosis of rat primary cultured hepatic stellate cells treated with platelet-derived growth factor BB (PDGF-BB).

METHODSHepatic stellate cells were cultured and treated with PDGF-BB+HGF, HGF or nothing (controls). Apoptosis of these hepatic stellate cells was evaluated by AO/EB staining, TUNEL and flow cytometry. Expression level of P65 was observed with immunocytochemical staining; DNA-protein binding complex of NF-kappa B was detected by electrophoretic mobility shift assay.

RESULTSThe cell apoptosis rate of the control group was lower than that of the PDGF-BB+HGF group. The apoptosis rate of the PDGF-BB+HGF group was lower than that of the HGF treated group; the expression of P65 was lower in the PDGF-BB+HGF group and HGF treated group compared to the normal control group; DNA-protein binding activity of NF-kappa B was respectively attenuated in the normal control group, PDGF-BB+HGF treated group and HGF treated group (P less than 0.05).

CONCLUSIONHGF can induce HSC apoptosis. Its possible mechanism may involve inhibiting DNA-protein binding activity of NF-kappa B and down-regulating the expression level of P65.

Animals ; Apoptosis ; drug effects ; Hepatic Stellate Cells ; drug effects ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; Male ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; metabolism

BACKGROUNDIn a suitable microenvironment, bone marrow mesenchymal stem cells (BMSCs) can transdifferentiate into myocardial cells whose special gene can be expressed as structural proteins. Growth factor (GF) plays an important role in the cell migration, survival and differentiation. However, the effect of GF on the cellular differentiation is not well understood. In this study, the hepatocyte growth factor (HGF) and insulin like growth factor-1 (IGF-1) were used in the mixed culture of BMSCs and myocardial cells and the effects of these growth factors on the GATA-4 expression of BMSCs were investigated.

METHODSBMSCs were isolated from the marrow of rabbit femurs and tibias and foetal rabbit ventricular myocytes were isolated with trypsin sequential digestion. These two kinds of cells were cocultured in a ratio of 1:1 for 6 weeks; cocultured cells with added HGF and IGF-1 were the experimental group. The differentiated BMSCs were collected using the laser capture, microdissection system and their RNA isolated. Immunocytochemical staining, transmission electron microscopy and reverse transcription-polymerase chain reaction were used to evaluate the transformation of the stem cells into cardiomyocytes like cells.

RESULTSWhen cultured separately, BMSCs did not express alpha-actin and the stem cells had many nucleoli. However, when cocultured with cardiomyocytes, BMSCs expressed alpha-actin and the cardiac transcription factor GATA-4 and showed cardiomyocyte like ultrastructure. In comparison with the control group, the experimental group exhibited the enhanced expression level of GATA-4. The GATA-4 expression of BMSCs increased gradually following the addition of HGF and IGF-1, reached the maximal level after two weeks and decreased slightly thereafter.

CONCLUSIONSBMSCs can transdifferentiate into cardiomyocytes like cells and express the cardiac transcription factor GATA-4 after being cocultured with myocardial cells. HGF and IGF-1 can stimulate transdifferentiation of BMSCs into cardiac phenotype and enhance the expression of GATA-4. These results indicate that growth factors have a great potential in clinical cellular therapy.

Animals ; Cell Differentiation ; drug effects ; Coculture Techniques ; GATA4 Transcription Factor ; genetics ; Hepatocyte Growth Factor ; pharmacology ; Insulin-Like Growth Factor I ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Rabbits

OBJECTIVE: To explore the effect of hepatocyte growth factor (HGF) on oxygen-glucose deprived injury and apoptosis of astrocytes. METHODS: The injury of primary cultured rat cerebral cortical astrocytes was induced by oxygen-glucose deprivation. Astrocytes were treated with HGF at various final concentrations of 20 - 100 ng/mL. The cell damage and viability were evaluated by the lactate dehydrogenase (LDH) released rate and the 3- (4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion method. Detection of apoptotic cells was determined by the flow cytometry, and the ultrastructure was observed by the transmission electron microscope. RESULTS: Oxygen-glucose deprivation increased the LDH release rate, decreased the cell viability and increased the number of apoptotic astrocytes. While exposed to HGF at the same condition, the LDH release rate decreased, the cell viability increased, and the percentage of apoptotic cells decreased (P <0.05). The maximum protective effect of HGF was observed at 60 ng/mL. CONCLUSION HGF can protect cultured astrocytes from oxygen-glucose deprived injury, and attenuate the apoptosis of astrocytes in a dose-dependent manner.
Animals ; Apoptosis ; drug effects ; Astrocytes ; pathology ; Cell Hypoxia ; Glucose ; pharmacology ; Hepatocyte Growth Factor ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism

AIMTo elucidate the effect of sphingosine kinase (SPK) on the hepatocyte growth factor (HGF)-induced migration of endothelial cells.

METHODSWe constructed recombinant adenoviral vectors, which contain SPK gene and its mutant respectively. These adenoviral vectors were packaged and amplified in 293 cells. And intracellular SPK activity was assayed via measurement of [32]P radioisotope labeled S1P; the effect of SPK activation on HGF-induced migration of endothelial cell was observed by Transwell technique.

RESULTSAdenoviral mediated expression of SPK gene increased in ECV 304 cells intracellular SPK activity, which in turn enhanced the HGF-induced migration. Whereas these activities were blocked by the dominant negative SPK gene.

CONCLUSIONThese findings show that SPK activation plays important roles in the regulation of HGF-induced migration of endothelial cells.

Adenoviridae ; metabolism ; Cell Line ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells.

METHODS(1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (I, cultured with serum-free DMEM containing 1 x 10(-7) mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI), ADSC-CM without insulin stimulation group (AC), insulin group (I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method (with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test.

RESULTS(1) Compared with those in C group [(287 +/- 47), (577 +/- 84) pg/mL, respectively], the secretion levels of VEGF and HGF in I group [(643 +/- 64), (930 +/- 68) pg/mL, respectively] were significantly increased (with t value respectively 18.869, 18.475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0.847 +/- 0.042, 0.798 +/- 0.022, respectively, which were higher than that in I and BC groups [0.665 +/- 0.028 (with t value respectively 4.579, 3.732), 0.674 +/- 0.031 (with t value respectively 3.761, 4.073), P values all below 0.01], and that in AI group was higher than that in AC group (t = 2.576, P < 0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8 +/- 1.9)%, (9.0 +/- 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 +/- 6.0)% (with t value respectively 12.891, 10.417), (31.4 +/- 7.4)% (with t value respectively 11.474, 9.783), P values all below 0.05 ], and that in AC group was higher than that in AI group (t = 8.548, P < 0.05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group (with t value respectively 4.076, 4.573, P values all below 0.05).

CONCLUSIONSParacrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.

Adipocytes ; cytology ; secretion ; Adipose Tissue ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Hepatocyte Growth Factor ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; secretion ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the mechanism by which exendin-4 promotes paracrine secretion of cytokines by adipose-derived stem cells (ADSCs).

METHODSIn vitro cultured SD rat ADSCs (fourth passage) with or without exendin-4 treatment underwent flow cytometry to characterize the surface markers. MTT assay was performed to assess the proliferation of the cells exposed to different concentrations (0-20 nm/L) of exendin-4, and the paracrine secretion of cytokines (bFGF, VEGF, HGF, and IGF-1) by the ADSCs was evaluated by qPCR. The changes in the expressions of p-Akt in the cells were analyzed by Western blotting and qPCR in response to exendin-4 (10 nm/L) with or without exposure to PI3K/Akt inhibitor LY-294002 (50 nm/L); bFGF, VEGF, HGF, and IGF-1 production in the cells were detected using ELISA kits.

RESULTSTreatment with exendin-4 for 12 h did not affect the surface marker profile of the ADSCs but promoted the cell proliferation (P<0.05). Exendin-4 significantly increased the mRNA expressions of VEGF, bFGF, HGF, and IGF-1 in a concentration-dependent manner, and 10 nm/L was the optimum concentration (P<0.05). Exendin-4 treatment resulted in significantly increased p-Akt expressions in the ADSCs, and PI3K/Akt inhibitor not only reversed such effects of exendin-4 on p-Akt but also diminished the exendin-4- mediated up-regulation of the paracrine cytokines.

CONCLUSIONExendin-4 can concentration-dependently promote the proliferative and paracrine capacities of ADSCs partially through the PI3K/Akt signaling pathway without affecting the surface marker profile of the cells.

Adipocytes ; cytology ; Animals ; Cell Proliferation ; Cells, Cultured ; Chromones ; Fibroblast Growth Factor 2 ; metabolism ; Hepatocyte Growth Factor ; metabolism ; Insulin-Like Growth Factor I ; metabolism ; Morpholines ; Peptides ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stem Cells ; cytology ; Up-Regulation ; Vascular Endothelial Growth Factor A ; metabolism ; Venoms ; pharmacology

OBJECTIVETo construct a tetracycline-inducible eukaryotic expression vector containing human hepatocyte growth factor (HGF) cDNA.

METHODSHuman HGF cDNA fragment was obtained by PCR from pUC-SRalpha/HGF plasmid and inserted into the restriction site between Mlu I and Sal I of the tetracycline-inducible eukaryotic expression vector pBI-L. pBI-L-HGF was constructed by DNA recombination in vitro, and was identified by restriction endonucleases digestion and sequencing.

RESULTSThe fragment of pBI-L-HGF digested with restriction endonucleases well corresponded to expectation, and the sequence of inserted HGF cDNA was correct according to the GenBank.

CONCLUSIONThe tetracycline-inducible eukaryotic expression vector of human HGF pBI-L-HGF has been constructed successfully, which allows further study of HGF gene therapy with much safety and easy manipulation.

DNA, Complementary ; genetics ; Eukaryotic Cells ; cytology ; metabolism ; Gene Expression ; drug effects ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Tetracycline ; pharmacology