Animals ; Humans ; Leptin ; genetics ; physiology ; Liver Cirrhosis ; etiology ; RNA, Messenger ; analysis ; Receptors, Cell Surface ; analysis ; physiology ; Receptors, Leptin ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

OBJECTIVESTo observe the expression of betaig-h3 in normal cornea and keratoconus and to elucidate the role of extracellular matrix in keratoconus.

METHODSIn situ hybridization was used to detect the expression of betaig-h3 in the cornea. The cDNA library was screened with human betaig-h3 cDNA probe to locate betaig-h3 mRNA in cells.

RESULTSExpression of betaig-h3 was found mainly in the stroma of the normal cornea and keratoconus, but decrease depending on the degree of keratopathy. In some serious cases, no expression signal was detected. The strongest expression was seen at the border of the normal region and keratoconus.

CONCLUSIONSbetaig-h3, the structural component of the extracellular matrix, can affect cell adhensiveness in the development of corneal fibrous interstitial organization. During the development of keratoconus, decreasing levels of betaig-h3 cause the diminution of corneal steadiness, which is related to formation of keratoconus.

Cornea ; metabolism ; Extracellular Matrix Proteins ; Humans ; Keratoconus ; metabolism ; Neoplasm Proteins ; genetics ; RNA, Messenger ; analysis ; Transforming Growth Factor beta ; Wound Healing

OBJECTIVETo explore the expressions of transforming growth factor-beta1 (TGF-beta1) and its signaling transduction molecule Smad 2 and their significance in the development of glomerulosclerosis.

METHODSUsing in situ hybridization and immunohistochemistry to detect Smad 2 mRNA expression and TGF-beta1, collagen IV, fibronectin expression in renal biopsies from 61 cases with a spectrum of glomerulonephritis including IgA nephropathy (40 cases), membranous glomerulonephritis (10 cases) and sclerosing glomerulonephritis (11 cases), compared with 11 cases of glomerular mild lesion with image analysis system.

RESULTSWith the exception of Smad 2 mRNA expression in mild type IgA nephropathy, all other types of diseased glomeruli showed increased expression of both TGF-beta1 and Smad 2 mRNA when compared with the 11 cases of mild glomerular lesions. The expressions of glomerular TGF-beta1 and Smad 2 mRNA positively correlated with collagen IV and fibronectin deposition in the glomeruli.

CONCLUSIONSTGF-beta1 and Smad 2 may be involved in the excessive deposition of glomerular extracellular matrix and play an important role in the development of glomerulosclerosis.

Collagen Type IV ; analysis ; DNA-Binding Proteins ; genetics ; Fibronectins ; analysis ; Glomerulonephritis ; metabolism ; Humans ; Immunohistochemistry ; Kidney Glomerulus ; chemistry ; RNA, Messenger ; analysis ; Smad2 Protein ; Trans-Activators ; genetics ; Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta1

OBJECTIVETo elucidate the effects of exogenous basic fibroblast growth factor (bFGF) on biological characteristics of rat osteoblasts cultured in vitro.

METHODSThe osteoblasts isolated from a Sprague-Dawley rat and cultured in vitro were treated with different concentrations of bFGF (5-50 ng/ml) respectively. At 24 hours after treatment, the proliferating cell nuclear antigen was measured with immunocytochemistry, alkaline phosphatase (ALP) activity was determined and the expression of transforming growth factor beta 1 (TGF-beta(1)) was detected to observe the effects of bFGF on growth and differentiation of osteoblasts.

RESULTSbFGF (5-50 ng/ml) could obviously promote the growth of osteoblasts. The intracellular expression of TGF-beta(1) mRNA increased significantly, but the intracellular ALP content decreased.

CONCLUSIONSbFGF can obviously stimulate the proliferation of osteoblasts and promote the synthesis of TGF-beta(1), but cannot promote the differentiation of osteoblasts.

Alkaline Phosphatase ; metabolism ; Animals ; Cells, Cultured ; Fibroblast Growth Factor 2 ; pharmacology ; Osteoblasts ; drug effects ; metabolism ; Proliferating Cell Nuclear Antigen ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

OBJECTIVETo study the effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis.

METHODSRT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TGFbetaRII recombinant plasmid which can be expressed in eukaryotic cells. Thirty-six male SD rats were randomly distributed into five groups: 10 in experimental liver fibrosis model induced by pig-serum as disease control group; 10 in antisense TGFbetaRII transfection as treatment group; 10 in pCDNA3 transfection as treatment control group and 6 in normal control group. The recombinant plasmid and empty vector (pCDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model respectively. Expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR and Western blot. We also tested ELISA of serum TGF-beta1, the contents of hepatic hydroxyproline, immunohistochemistry of type I and III collagen, and VG staining for pathological study.

RESULTSThe antisense TGFbetaRII expressing plasmid could be well expressed in vivo, and could block the mRNA and protein expression of TGFbetaRII in the fibrotic liver induced by pig serum. Its expression also reduced the level of TGF-beta1 [antisense treatment group (23.16+/-3.13) ng/ml, disease control group (32.96+/-3.79) ng/ml; F=36.73, 0.01]. Compared with the disease control group, the contents of hepatic hydroxyproline [antisense treatment group (0.17+/-0.01) mg/g liver, disease control group (0.30+/-0.03) mg/g liver; F=15.48, 0.01] and the deposition of collagens type I and type III decreased in the antisense group (antisense treatment group collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F values are 69.87, 70.46, 0.01). And its expression also improved the pathologic classification of liver fibrosis models (0.01).

CONCLUSIONThe results demonstrate that TGF-beta plays a key role in liver fibrogenesis and the prevention of liver fibrosis by antisense TGFbetaRII recombinant plasmid intervention may be therapeutically useful.

Animals ; Antisense Elements (Genetics) ; therapeutic use ; Liver Cirrhosis, Experimental ; etiology ; therapy ; Male ; Plasmids ; therapeutic use ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; antagonists & inhibitors ; genetics ; Transforming Growth Factor beta ; physiology

BACKGROUND/AIMS: The genetic polymorphism of transforming growth factor-beta1 (TGF-beta1) at codons 10 and 25 which influences the production of TGF-beta1 is related to fibrogenesis in the lung and liver. We evaluated the genetic polymorphism at codons 10 and 25 in controls and in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC). METHODS: Blood samples were collected from controls (n=35), patients with LC (n=64), and HCC (n=49). Genomic DNA was isolated and polymerase chain reaction (PCR) was done for a segment including codons 10 and 25. The results of direct sequencing for PCR products were compared between the controls and the patients. RESULTS: There was no genetic polymorphism at codon 25 and three types of genetic polymorphism at codon 10. The leucine homozygous genotype (CTG/CTG) at codon 10 was more common in patients with LC than the controls (p=0.01) and especially in patients with LC caused by HBV (p=0.004). The polymorphism at codons 10 in patients with HCC was similar to the controls. However, leucine homozygous genotype was more common in patients with HCC of uninodular morphology than those of massive morphology (p=0.007). CONCLUSIONS: The genetic polymorphism of TGF-beta1 at codon 10 might be associated with LC and morphology of HCC. The potential usefulness of TGF-beta1 genotyping needs further studies in large scale.
Adult ; Aged ; Carcinoma, Hepatocellular/*genetics ; Codon/genetics ; Female ; Genotype ; Humans ; Korea ; Liver Cirrhosis/*genetics ; Liver Neoplasms/*genetics ; Male ; Middle Aged ; *Polymorphism, Genetic ; Sequence Analysis, Protein ; Transforming Growth Factor beta/*genetics ; Transforming Growth Factor beta1

Activin Receptors, Type I ; analysis ; Animals ; Arcuate Nucleus of Hypothalamus ; chemistry ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Immunohistochemistry ; Liver Regeneration ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; analysis ; Rats ; Receptors, Transforming Growth Factor beta ; analysis ; Sodium Glutamate ; Transforming Growth Factor alpha ; analysis ; genetics ; Transforming Growth Factor beta ; analysis ; genetics

BACKGROUNDNanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects.

METHODSTwenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation.

RESULTSThe tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone formation rate and antibending strength of group A was significantly higher than those of group B and C. The defects in blank control were not healed.

CONCLUSIONSThe hBMP2 gene activated nanobone putty exhibited osteoinductive ability, and had a better bone defect repair capability than that of nanobone putty only.

Absorbable Implants ; Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; analysis ; genetics ; Female ; Male ; Mice ; Osteogenesis ; physiology ; Rabbits ; Transforming Growth Factor beta ; analysis ; genetics

OBJECTIVESTo investigate the biological responses of cultured hepatic stellate cells (HSC) at different differentiated stages on exotic transforming growth factor (TGF-beta1).

METHODSHSC was isolated from rat and primarily cultured in uncoated disc for 1 d, 4 d and 7 d, when the cells were at quiescent, intermediate activated and full activated stages respectively. The cells were incubated with 10 pmol/L to 500 pmol/L TGF-beta1 for 24 h, cell proliferation was measured with [3H] TdR incorporation, alpha-smooth muscle actin (alpha-SMA) and type I collagen protein were assayed with Western blot, and total protein secretion in the culture supernatant was analyzed by [3H] proline pulse and collagenase digestion. HSC was treated with 100 pmol/L TGF- beta1 for 15 min to 90 min, and type I pro-collagen mRNA level was assayed by Northern blot.

RESULTSTGF-beta1 remarkably inhibited d1 HSC proliferation, the percentage of [3H] TdR incorporation at 10 pmol/L to 500 pmol/L TGF-beta1 was 52.8% to 16.8% of the control, q value was 5.44 to 10.37 and P<0.01 vs control. But TGF-beta1 had no influence on d4 and d7 HSC. As the cells cultivation prolonged and activated, the basal levels of alpha-SMA, type I collagen and gene expression increased gradually. TGF-beta1 increased the above protein and gene expression. The basal and TGF-beta1 stimulated total protein secretion levels at d1-d7 HSC were 804+/-274 vs 1200+/-708; 2966+/-1701 vs 6160+/-1123, t=3.84, P<0.01; 2580+/-767 vs 4583+/-1467, t=2.96, P<0.05. While d4 HSC showed the strongest response of total protein secretion and alpha-SMA expression.

CONCLUSIONSTGF-beta1 remarkably inhibited quiescent HSC proliferation, and promoted HSC collagen production at both quiescent and activated HSC. Intermediate HSC had the strongest response to TGF-beta1, while activated HSC lost the response to TGF-beta1 inhibitory growth, and TGF-beta1 exerted divergent actions on HSC as the cells activated.

Activin Receptors, Type I ; analysis ; Animals ; Cell Division ; drug effects ; Collagen Type I ; genetics ; Liver ; cytology ; drug effects ; metabolism ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factor beta1

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; Corneal Injuries ; Fibronectins ; analysis ; Immediate-Early Proteins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Rabbits ; Signal Transduction ; physiology ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; analysis ; genetics ; physiology ; Transforming Growth Factor beta1 ; Wound Healing ; physiology