OBJECTIVETo investigate the expression of bFGF, ET-1 and SCF in different passages of cultured dermal papilla cells (DPC), and their possible effect on biological behaviour of DPC.

METHODSThe expression of bFGF, ET-1 and SCF in different passages of cultured DPC was detected by immunocytochemistry and in situ hybridization.

RESULTThe expression of ET-1 and SCF in early passages of cultured DPC was stronger, but became negative in late passages (>6 passages). The stronger the expression of ET-1 and SCF in DPC, the higher ability of DPC to induce hair follicle regeneration.

CONCLUSIONThe expression strength of ET-1 and SCF is related to the ability of DPC inducing hair follicle regeneration.

Endothelin-1 ; analysis ; genetics ; Fibroblast Growth Factor 2 ; analysis ; genetics ; Hair Follicle ; chemistry ; cytology ; physiology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Stem Cell Factor ; analysis ; genetics

Cytokines have pleiotropic regulatory effects on hematopoietic cells and many other cell types that participate in host defence and repair processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages and regulates the biological functions expressed by mature cells of these lineages. Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. In order to determine the complementary DNA (cDNA) of canine GM-CSF and canine SCF, cDNA clones were generated from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) and bone marrow cells by reverse transcription PCR amplification. The canine GM-CSF cDNA obtained in this study contains an open reading frame encoding 144 amino acid residues and has 53-75% homology with those of human, cat, sheep, pig, cow and mouse, Canine SCF cDNA consist of an open reading frame encoding 274 amino acid residues and shares 81-92% homology with those of human, cat, pig, cow and mouse.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; Cattle ; *Cloning, Molecular ; Codon ; DNA, Complementary/analysis/*genetics ; Dogs/blood/*genetics ; Gametogenesis ; Gene Amplification ; Granulocyte-Macrophage Colony-Stimulating Factor/chemistry/*genetics ; Hematopoiesis ; Humans ; Mice ; Molecular Sequence Data ; Open Reading Frames ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sheep ; Stem Cell Factor/chemistry/*genetics ; Swine

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

Stem cell factor is an important hematopoietic growth factor. In this study, the human stem cell factor was produced by recombinant E. coli, and the structure and biological activity of the recombinant stem cell factor(rhSCF) was studied. It was indicated that the rhSCF was a uncovalent dimer in phosphate buffer,and had the correct mass spectra, mass peptides spectra, composition of amino acid, N-terminal sequernce, C-terminal sequence and intrachain disulfide linkages, rhSCF alone or synergy with rhG-CSF could mobilze hematopoietic progenitors to blood in monkey.
Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chromatography, High Pressure Liquid ; Haplorhini ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; pharmacology ; Sequence Analysis, Protein ; Spectrometry, Mass, Fast Atom Bombardment ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stem Cell Factor ; chemistry ; genetics ; metabolism ; pharmacology

OBJECTIVETo study the expression of hHSF in E. coli and its effect on the mobilization of hematopoietic stem/progenitor cells.

METHODSThe hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E. coli BL21(DE3) and expressed with IPTG induction. Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding. Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy. The N terminal amino acid sequence rhHSF was determined by protein sequencing. rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells. Eight rhesus monkeys were equally divided into two groups. The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF.

RESULTSThe sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins. The purity of target protein was over 95%. The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540. The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF. The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively.

CONCLUSIONhHSF is highly expressed in E. coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys. hHSF shows distinct synergistic effect with G-CSF.

Animals ; Chemokine CXCL2 ; chemistry ; genetics ; pharmacology ; Escherichia coli ; genetics ; Female ; Genetic Vectors ; genetics ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Macaca mulatta ; Male ; Protein Folding ; Recombinant Proteins ; chemistry ; pharmacology ; Sequence Analysis, Protein ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODSThe HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTSHFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONHFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Cell Adhesion ; Cell Cycle ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; metabolism ; Epidermis ; Fetus ; G1 Phase ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Keratinocytes ; cytology ; Keratins ; analysis ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Microscopy, Fluorescence ; Plasmids ; genetics ; Stem Cells ; chemistry ; cytology ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the mechanism that the formulas for activating blood and resolving stasis can regulate hemopoietic stem cell to produce new blood.

METHODRats were established animal model of acute cerebral infarction by referencing Olivette' method. They were randomly divided into model group, the group of the high, middle, low dose of the formulas for activating blood and resolving stasis. Each group and then wasrandomly divided into subgroups by 1, 3, 7, 14, 28 d. Xuesaitong capsule was formulated into 20, 40, 60 g x L(-1) with normal saline. The rats were given gavage drugs once a day until the experient ended, and the model group was administrated by intragastrical perfusion of normal saline. ELISA was used to detect the expression of SCF in peripheral blood and bone marrow among different groups at different time points. Flow cytometry was used to observe the changes of CD117 in blood and bone marrow.

RESULTThe CD117+ HSC and SCF concentration in peripheral blood and bone marrow of model group were increasing during 1-14 d,there was a peak on the 14th day, then the expression was reducing. CD117+ HSC and SCF concentration rising trend in the group of the high, middle dose of the formulas for activating blood and resolving stasis was preceded model group (P < 0.05).

CONCLUSIONActivating blood and resolving stasis can regulate hemopoietic stem cell to produce new blood, and it is through the regulation of CD117+ HSC number to achieve the purpose.

Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Capsules ; Cerebral Infarction ; blood ; drug therapy ; genetics ; metabolism ; Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; administration & dosage ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Humans ; Male ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cell Factor ; genetics ; metabolism

To investigate the expression and function of the receptors of early-acting cytokines on cord blood CD34(+) hematopoietic stem/progenitor cells, the studies of Flt3 and c-kit were undertaken at the gene and protein levels. Fresh and cultured cord blood CD34(+) stem/progenitor cells were analyzed by flow cytometric two-color direct immunofluorescence methods and RT-PCR, while function of receptors was studied in vitro. It was found that (68.8 +/- 15.4)% of CD34(+) cells expressed Flt3, (50.6 +/- 12.7)% of CD34(+) cells expressed c-kit, the proportion of CD34(+) cells expressing Flt3 and c-kit decreased as in vitro culture time extended. It was concluded that cord blood CD34(+) stem/progenitor cells are capable of expressing Flt3 and c-kit for as long as 2 - 3 weeks in liquid medium, during the first week of culture, SCF and FL enhanced the generation of cells and progenitors notably.
Antigens, CD34 ; blood ; Cells, Cultured ; Fetal Blood ; cytology ; Flow Cytometry ; Hematopoietic Stem Cells ; chemistry ; Humans ; Proto-Oncogene Proteins ; analysis ; genetics ; physiology ; Proto-Oncogene Proteins c-kit ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptor Protein-Tyrosine Kinases ; analysis ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; fms-Like Tyrosine Kinase 3

AIMTo study the effect of catechin, the active component of Spatholobus suberectus Dunn, on bone marrow cell cycle and the expression of IL-6 and GM-CSF mRNA in spleen cells of normal and marrow-depressed mice in order to clarify the mechanism of hematopoietic-supportive effect of catechin.

METHODSFlow cytometry was adopted to investigate the influence of catechin on bone marrow cell cycle in mice and the expression of IL-6 and GM-CSF mRNA induced by catechin in spleen cells was detected by RT-PCR technique simultaneously.

RESULTSThe cell proportion of normal and marrow-depressed mice in G0/G1 phase was reduced significantly, while that in S + G2/M phase increased significantly. Being induced by catechin, IL-6 mRNA and GM-CSF mRNA in spleen cells were markedly up-regulated.

CONCLUSIONCatechin (2 g x L(-1), intraperitoneally injected to mice daily immediately after irradiation for 7 consecutive days) was shown to promote the expression of IL-6 mRNA and GM-CSF mRNA in spleen cells of mice, through which it can accelerate bone marrow cells of normal mice into cell cycle and help those of marrow-depressed mice to get out of "G1-phase-block", enter into cell cycle and radically accelerate the proliferation and differentiation of hematopoietic stem cell/hematopoietic progenitor cell (HSC/HPC).

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Catechin ; isolation & purification ; pharmacology ; Cell Cycle ; drug effects ; Fabaceae ; chemistry ; Female ; Gene Expression Regulation ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Hematopoietic Stem Cells ; cytology ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Mice ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Spleen ; cytology ; metabolism

BACKGROUNDHuman umbilical cord blood contains an abundance of immature stem/progenitor cells, which may participate in the repair of hearts that have been damaged by myocardial infarction (MI). This study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (hUCBC) transplantation on cardiac function and left ventricular remodeling in rat model of MI.

METHODSForty-five male Wistar rats were randomized into three groups: MI or control group (n = 15), MI plus cell transplantation (n = 15), and sham group (n = 15). Acute myocardial infarction (AMI) was established by ligating the left anterior descending artery, thereafter, hUCBC were implanted into the marginal area of infarcted myocardium. In MI/control group, DMEM was injected instead of hUCBC following the same protocol. Left ventricular function assessment was carried out by echocardiography and invasive hemodynamic measurements one month post MI. All rats were sacrificed for histological and immunochemical examinations.

RESULTSThe transplanted hUCBC survived and engaged in the process of myocardial repair in the host heart. Echocardiography demonstrated that left ventricular function improved significantly in the rats that underwent cell transplantation. Hemodynamic studies found a significantly decreased left ventricular end-diastolic pressure (LVEDP) [(21.08 +/- 8.10) mmHg vs (30.82 +/- 9.59) mmHg, P < 0.05], increase in +dp/dt(max) [(4.29 +/- 1.27) mmHg/ms vs (3.24 +/- 0.75) mmHg/ms, P < 0.05), and increase in -dp/dt(max) [(3.71 +/- 0.79) mmHg/ms vs (3.00 +/- 0.49) mmHg/ms, P < 0.05] among MI group with hUCBC transplantation when compared with MI/control group. Masson's trichrome staining revealed that the collagen density in the left ventricle was significantly lower in rats of transplantation group than that in the MI control groups [(6.33 +/- 2.69)% vs (11.10 +/- 3.75)%, P < 0.01]. Based on immunostaining of alpha-actin, the numbers of microvessels were significantly (P < 0.01) increased at the boundary of infarction site. Similarly higher mRNA expression of vascular endothelial growth factor (VEGF) 164 and VEGF188 were found at 7- and 28-day post cell transplantation in MI group with hUCBC transplantation when compared with MI/control group.

CONCLUSIONSTransplanted hUCBC can survive in host myocardium without immunorejection, significantly improve left ventricular remodeling after AMI and promote a higher level of angiogenesis in the infarct zones. All these factors beneficially affect cardiac repair in the setting of MI. Therefore human umbilical cord blood may be potential source for cell-based therapy for AMI.

Actins ; analysis ; Animals ; Antigens, CD34 ; analysis ; Collagen ; metabolism ; Cord Blood Stem Cell Transplantation ; Electrocardiography ; Gene Expression ; Humans ; Immunohistochemistry ; Leukocytes, Mononuclear ; chemistry ; cytology ; transplantation ; Male ; Myocardial Infarction ; physiopathology ; surgery ; Myocardium ; chemistry ; metabolism ; pathology ; Neovascularization, Physiologic ; physiology ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous ; Vascular Endothelial Growth Factor A ; genetics ; Ventricular Function, Left ; physiology