Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.
Animals ; Apoptosis ; Body Patterning ; Bone Morphogenetic Proteins/biosynthesis* ; Developmental Biology ; Ectoderm/metabolism* ; Extremities/embryology* ; Fibroblast Growth Factor 10/metabolism* ; Fibroblast Growth Factors/biosynthesis* ; Gene Expression Regulation ; Hedgehog Proteins/biosynthesis* ; Homeodomain Proteins/biosynthesis* ; Mesoderm/metabolism* ; Mice ; Signal Transduction ; Wnt Proteins/biosynthesis*

Fibroblast growth factor 23 (FGF23) is a hormone that is mainly secreted by osteocytes and osteoblasts in bone. The critical role of FGF23 in mineral ion homeostasis was first identified in human genetic and acquired rachitic diseases and has been further characterised in animal models. Recent studies have revealed that the levels of FGF23 increase significantly at the very early stages of chronic kidney disease (CKD) and may play a critical role in mineral ion disorders and bone metabolism in these patients. Our recent publications have also shown that FGF23 and its cofactor, Klotho, may play an independent role in directly regulating bone mineralisation instead of producing a systematic effect. In this review, we will discuss the new role of FGF23 in bone mineralisation and the pathophysiology of CKD-related bone disorders.
Calcification, Physiologic ; Fibroblast Growth Factors ; biosynthesis ; metabolism ; physiology ; Glucuronidase ; metabolism ; Humans

Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.
Animals ; Bacteria ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; Disease Models, Animal ; Fibroblast Growth Factors ; biosynthesis ; Genetic Vectors ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; isolation & purification ; Inclusion Bodies ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis

Crystallography, X-Ray ; Escherichia coli ; metabolism ; Fibroblast Growth Factors ; chemistry ; genetics ; metabolism ; Heparin ; metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Isoforms ; chemistry ; metabolism ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Sulfates ; chemistry ; metabolism

The aim of the study is to construct two vectors for efficient expression of soluble recombinant proteins. The first vector was constructed by cloning the HisSUMO fragment into an expression vector pET30a(+) to fuse with the gene of interest (designated as HisSUMO Express). The second vector was constructed in the same way, but with a hydroxylamine cleavage site between HisSUMO and the gene of interest for an economic purpose (designated as HisSUMO Economic). The mouse fibroblast growth factor-21(mFGF-21), which was difficult to express in routine-used expression vectors, was taken as an example to test the vectors. The results showed that the mFGF-21 was expressed at high level in both vectors. The Sumo/mFGF-21 fusion protein accounted for more than 40% of the total bacterial protein. The fusion protein was purified with Ni-TNA column, and the HisSUMO was removed by cleavage of the fusion protein with either hydroxylamine solution or SUMO protease I. The concentration of the purified mFGF-21 mature protein was 54 mg/L and the recovery rate was 6%. The purified proteins derived from either hydroxylamine or SUMO protease I cleavage could stimulate glucose up-take by adipocytes. These results indicated that both HisSUMO Express and HisSUMO Economic were useful expression vectors for efficient expression of soluble recombinant proteins.
Animals ; Fibroblast Growth Factors ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Hydroxylamine ; chemistry ; Mice ; Peptide Hydrolases ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Solubility

Primary HLSCs were successfully cultured and assayed by AE5 in vitro. Constructed eukaryotic expressive vector of pEGFP-bFGF was transferred into the human limbal stem cells by the liposome-mediated technique, and 48 hours later, specific green fluorescence was observed by fluorescence microscope. The gene transfeetion efficiency was 20%-30%. Then the model of cells injury was created by use of NaOH. The cells were divided into four groups: Normal, bFGF, NaOH and bFGF+NaOH. The cellular viability in each group was measured by MTT colorimetry, and the cellular apoptosis rate and necrosis rate were observed by laser scanning confocal microscopy. The cellular viability in bFGF+NaOH group was higher than that in NaOH group (P < 0.05) ,while the cellular apoptosis rate plus necrosis rate displayed significant difference between the two groups (P < 0.05). The pEGFP-bbGF gene was noted to be successfully transferred into HLSCs and the cells were found growing well. These indicated that bFGF gene has a protective effect on the HLSCs injured by NaOH. We have also probed the feasibility of trying the treatment for ocular surface disease through gene engineering recombined tissue engineering.
Cells, Cultured ; Cornea ; cytology ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Gene Expression ; Genetic Therapy ; methods ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cells ; cytology ; Tissue Engineering ; Transfection ; Vascular Endothelial Growth Factors ; genetics ; metabolism

OBJECTIVETo observe the expression pattern of albumin during the hepatocyte differentiation by human bone marrow stem cells in vitro.

METHODSHuman bone marrow cells were harvested and cultured in the presence of hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and lymphocyte inhibitory factor (LIF). Cells were stained immunohistochemically by albumin specific antibody and examined under a confocal microscope. Supernatant albumin level was measured biochemically on a serial time points of the culture.

RESULTSBy this condition, the attached cells became mature morphologically in 1 week of culture. Hepatocyte-specific albumin could be detected in mature cells. The albumin level revealed a time-dependent change during a 4-week culture.

CONCLUSIONHuman bone marrow cells could be induced to differentiate to mature hepatocytes that produce and secret albumin in vitro. These cells may contribute to a stable source of hepatocytes for clinical hepatocyte transplantation and artificial liver support system.

Albumins ; biosynthesis ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Fibroblast Growth Factors ; pharmacology ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology

OBJECTIVETo detect the expression of basic fibroblast growth factor (bFGF) in human ocular tissues, and to assess the effect of bFGF on the proliferation of human cataract lens epithelial cells (LECs) and its correlation with age.

METHODSEnucleated eyes were subjected to immunostaining for bFGF protein. Human cataract LECs were cultured in vitro, and treated with bFGF for 48 hr. Proliferation was estimated by the positive area ratio of proliferating cell nuclear antigen (PCNA) in immunohistochemistry.

RESULTSbFGF protein was found in various human ocular tissues. bFGF stimulated human cataract LEC proliferation, and there was an age-related decrease in responsiveness of human cataract LECs to bFGF (P < 0.05).

CONCLUSIONbFGF might play an important role in the proliferation of residual human cataract LECs after cataract surgery.

Adolescent ; Adult ; Age Factors ; Cataract ; metabolism ; pathology ; Child ; Child, Preschool ; Epithelial Cells ; chemistry ; pathology ; Fibroblast Growth Factor 2 ; biosynthesis ; Humans ; Immunohistochemistry ; Infant ; Infant, Newborn ; Lens, Crystalline ; chemistry ; pathology ; Middle Aged ; Proliferating Cell Nuclear Antigen ; analysis

OBJECTIVETo investigate intratumoral microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BFGF) in hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE) and to evaluate their significance.

METHODSMVD and expression of VEGF and BFGF in cancerous tissues were examined in forty specimens resected from patients with HCC using immunohistochemical methods. Among these patients, 20 patients received 1 to 7 treatments of TACE prior to II-phase surgical resection (TACE group), the other 20 patients were treated by operation without receiving any other treatment preoperatively (surgical group). There was no significant difference in clinical features between the two groups. MVD was assessed by counting immunostained endothelial cells within a certain area, and staining intensity of VEGF was assessed quantitatively with computer-assisted image analyzer. The expression of BFGF was determined by cell-positive or cell-negative.

RESULTSThe average MVD was 130.51 75.5 in TACE group and 152.35 58.80 in surgical group. There was no significant difference between the two groups (t=-1.021, P=0.341). Staining intensity of VEGF was 645.60 543.27 in TACE group, higher than in surgical group (158.28 188.48, t=281, P<0.001). BFGF-positive rate was 35% in TACE group and 40% in surgical group. There was no significant difference (x(2)=0.107, P=0.744).

CONCLUSIONSThe results indicate that survived cancerous tissue has rich vascularity and the expression of VEGF of the cancerous cells can be enhanced by TACE which may play an important role in reestablishment of blood supply to tumor after TACE.

Carcinoma, Hepatocellular ; metabolism ; pathology ; physiopathology ; therapy ; Catheterization ; Embolization, Therapeutic ; Endothelial Growth Factors ; biosynthesis ; Fibroblast Growth Factor 2 ; biosynthesis ; Humans ; Liver Neoplasms ; metabolism ; pathology ; physiopathology ; therapy ; Lymphokines ; biosynthesis ; Neovascularization, Pathologic ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Leptin, the product of ob gene, is an endocrine hormone that regulates adipose tissue mass. Recently, leptin has been found to generate a growth signal involving a tyrosine kinase-dependent intracellular pathway and promote angiogenic processes via activation of leptin receptor (Ob-R) in endothelial cells. However, it is not clear how leptin functions to promote multi-step processes involved in the neovascularization at the atherosclerotic plaque. We have examined the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and Ob-R in human atherosclerotic lesions, leptin-mediated angiogenesis in vivo and in vitro. Immunohistochemical analysis of human atherosclerotic aorta revealed an increased expression of Ob-R in the intima of neorevascularized regions and of both MMPs and TIMPs predominantly in the endothelial lining of intimal neovessels and macrophages/foam cells. In the rat corneal angiogenesis assay, leptin elicited a comparable sensitivity of angiogenic activity to those of vascular endothelial growth factor (VEGF). The immunohistological analysis of the leptin-treated rat cornea showed definitive rises in Ob-R, MMPs and TIMPs expression as well as those of VEGF receptor (VEGFR-1). Leptin (10-40 ng/ml) induced proliferation of the human umbilical vein endothelial cells (HUVECs) and elevation of MMP-2, MMP-9, TIMP-1, and TIMP-2 expression in a dose-dependent manner. Leptin also induced increases of MMP-2, MMP-9, TIMP-1, and Up-regulated the human coronary artery smooth muscle cells (HCASMCs). These findings suggest that leptin, a hormone with pluralistic properties including a mitogenic activity on vascular endothelial cells, plays a role in matrix remodeling by regulating the expression of MMPs and TIMPs. Taken together, our findings further provide evidences for leptin's role as an angiogenesis inducer in the normal organ (rat cornea) and in aberrant vasculature under duress like atherosclerosis.
Animal ; Arteriosclerosis/metabolism ; Blotting, Western ; Cell Division ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Growth Factors/metabolism ; Endothelium, Vascular/*cytology/*enzymology ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2/metabolism ; Immunohistochemistry ; Leptin/*chemistry/metabolism/*physiology ; Lymphokines/metabolism ; Matrix Metalloproteinases/*biosynthesis ; *Neovascularization, Pathologic ; Rats ; Receptor Protein-Tyrosine Kinases/metabolism ; Receptors, Growth Factor/metabolism ; Recombinant Proteins/metabolism ; Support, Non-U.S. Gov't ; Tissue Inhibitor of Metalloproteinases/metabolism ; Umbilical Veins/metabolism ; Up-Regulation