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*

OBJECTIVETo express and purify the recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2m) in prokaryotic system and to develop highly-specific monoclonal antibodies.

METHODSAn engineered E. coli strain expressing rhBMP-2m was fermented. The bacterial cells were firstly lysed and then the rhBMP-2m inclusion bodies were isolated by centrifugation. After the inclusion bodies had been solubilized by high-concentration denaturing agents, denatured rhBMP-2m was purified by cation ion-exchange chromatography. Biologically active rhBMP-2m was obtained by refolding of purified denatured rhBMP-2m through direct dilution. The refolded rhBMP-2m was used to immunize Balb/c mice to develop anti-rhBMP-2m monoclonal antibodies using classic hybridoma technique.

RESULTSrhBMP-2m with a purity greater than 95% was obtained on reduced SDS-PAGE. The refolded rhBMP-2m was measured to be bioactive by the induction of alkaline phosphatase activity in MC3T3-E1 cells. Two hybridoma cell lines that stably secreted anti-rhBMP-2m antibody were developed from the immunized mice.

CONCLUSIONBioactive rhBMP-2m protein and its monoclonal antibodies were successfully prepared, which will provides a solid base for future studies on rhBMP-2.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Bone Morphogenetic Proteins ; biosynthesis ; immunology ; Escherichia coli ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C

OBJECTIVETo construct a recombinant adenovirus vector containing bone morphogenic protein-7 (BMP-7) gene and to identify its biological activities in proximal tubule epithelial cells (PTECs).

METHODSForty-six fragments of BMP-7 gene were obtained by PCR method and then were ligated to the full-length gene. The full length sequence of BMP-7 was subcloned into pBluescript II(+) vectors, and confirmed by sequencing. Double digested with Not I and Hind III, BMP-7 gene was inserted into pShuttle-CMV. EcoR I pre-linearized pShuttle plasmid was transformed into competence bacterium BJ5183 to obtain the recombinant adenovirus-BMP-7 by efficient homologous recombination. Then the AdBMP-7 was obtained by packaging Pac I linearized in 293 cells. Adenoviral titer was determined by adenovirus fluorescent detection kit. The protein expression of BMP-7 in PTECs was respectively detected by ELISA and Western blot. RT-PCR method was used for analyzing the alpha-smooth muscle action (alpha-SMA) expression in PTECs, which was treated consecutively with TGF-beta and AdBMP-7.

RESULTSThe recombinant plasmid AdBMP-7 was successfully generated, which increased BMP-7 protein expression levels in PTECs, and down-regulated TGF-beta-induced alpha-SMA expression.

CONCLUSIONThe bioactive recombinant adenovirus AdBMP-7 has been successfully constructed, which may be effective in inhibition of chronic renal fibrosis.

Adenoviridae ; genetics ; metabolism ; Animals ; Base Sequence ; Bone Morphogenetic Protein 7 ; biosynthesis ; genetics ; Cells, Cultured ; Epithelial Cells ; metabolism ; Genetic Vectors ; genetics ; Kidney Tubules, Proximal ; cytology ; metabolism ; Molecular Sequence Data ; Rats ; Recombinant Proteins ; biosynthesis ; genetics

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.
Animals ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; Mice ; RNA Interference ; Recombinant Proteins ; biosynthesis ; genetics ; Smad6 Protein ; biosynthesis ; genetics ; Transfection

Bone morphogenetic protein 2 (BMP2), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, is a multifunctional molecule with distinct abilities to induce bone formation. BMP2 has been identified to have eminent pharmaceutical importance for clinical application. We previously constructed stable cell line in Chinese hamster ovary cells (CHO) that highly expressed recombinant human BMP2 (rhBMP2). For large-scale production of the recombinant protein used in clinical application, it is critical to have both high expression and stability of the protein. In the present study, the stability of the cell line (rCHO(hBMP2)-C8) with the highest expression, as well as the stability of rhBMP2 protein were investigated systematically. We cultured the rCHO (hBMP2)-C8 cell line in the presence or absence of MTX for two months, the cell growth and rhBMP2 production characteristics were examined during the culture; we found the duration that the rCHO(hBMP2)-C8 cell line could secret rhBMP2 continually into the serum-free medium. Moreover, we detected the temperature sensitivity of rhBMP2 in culture medium. This study will contribute to our understanding for further producing rhBMP2 by large-scale culture technology.
Animals ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; genetics

BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.
Bone Morphogenetic Protein 6 ; biosynthesis ; genetics ; Carrier Proteins ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Solubility ; Transformation, Bacterial

To determine which genes are regulated by thyroid stimulating hormone (thyrotropin, TSH), insulin and insulin-like growth factor-1 (IGF-1) in the rat thyroid, we used the microarray technology and observed the changes in gene expression. The expressions of genes for bone morphogenetic protein 6, the glucagon receptor, and cyclin D1 were increased by both TSH and IGF-1; for cytochrome P450, 2c37, the expression was decreased by both. Genes for cholecystokinin, glucuronidase, beta, demethyl-Q 7, and cytochrome c oxidase, subunit VIIIa, were up-regulated; the genes for ribosomal protein L37 and ribosomal protein L4 were down-regulated by TSH and insulin. However, there was no gene observed to be regulated by all three: TSH, IGF-1, and insulin molecules studied. These findings suggest that TSH, IGF-1, and insulin stimulate different signal pathways, which can interact with one another to regulate the proliferation of thyrocytes, and thereby provide additional influence on the process of cellular proliferation.
Animals ; Bone Morphogenetic Protein 6 ; Bone Morphogenetic Proteins/biosynthesis ; Cell Line, Tumor ; Cyclin D1/biosynthesis ; *Gene Expression Profiling ; *Gene Expression Regulation ; Insulin/*biosynthesis/metabolism ; Insulin-Like Growth Factor I/*biosynthesis ; Models, Genetic ; *Oligonucleotide Array Sequence Analysis ; Rats ; Receptors, Glucagon/biosynthesis ; Thyroid Gland/*metabolism ; Thyrotropin/*biosynthesis/metabolism ; Time Factors

OBJECTIVETo evaluate the levels of bone morphogenetic protein-15 (BMP-15) in human follicular fluid (FF) and its association with response to ovarian stimulation.

METHODSWestern blotting was performed to determine the levels of BMP-15 in FF obtained from follicle aspirates in 70 patients undergoing IVF treatment. According to the response to ovarian stimulation the patients were divided into poor responder group and normal responder group.

RESULTBMP-15 levels in FF of poor responders were significantly higher than those in normal responders (1.01 +/- 0.34 vs 0.77 +/- 0.24, P<0.01).

CONCLUSIONIncreased levels of BMP-15 in FF may be associated with poor response to ovarian stimulation.

Adult ; Blotting, Western ; Bone Morphogenetic Protein 15 ; Female ; Follicle Stimulating Hormone ; administration & dosage ; Follicular Fluid ; drug effects ; metabolism ; Gonadotropin-Releasing Hormone ; administration & dosage ; analogs & derivatives ; Growth Differentiation Factor 9 ; Humans ; Infertility, Female ; metabolism ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Ovary ; drug effects ; metabolism ; Ovulation Induction

BACKGROUNDIcariine is a flavonoid isolated from a traditional Chinese medicine Epimedium pubescens and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis. But the mechanism is unclear. The aim of the study was to research the effect of Icariine on the proliferation and differentiation of human osteoblasts.

METHODSHuman osteoblasts were obtained by inducing human marrow mesenchymal stem cells (hMSCs) directionally and were cultured in the presence of various concentrations of Icariine. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of Icariine on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of calcified nodules were assayed to observe the effect on cell differentiation. The expression of bone morphogenetic protein 2 (BMP-2) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSIcariine (20 microg/ml) increased significantly the proliferation of human osteoblasts. And, Icariine (10 microg/ml and 20 microg/ml) increased the activity of ALP and the amount of calcified nodules of human osteoblasts significantly (P < 0.05). BMP-2 mRNA synthesis was elevated significantly in response to Icariine (20 microg/ml).

CONCLUSIONSIcariine has a direct stimulatory effect on the proliferation and differentiation of cultured human osteoblast cells in vitro, which may be mediated by increasing production of BMP-2 in osteoblasts.

Alkaline Phosphatase ; analysis ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Humans ; Osteoblasts ; cytology ; drug effects ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta ; biosynthesis ; genetics

Bone morphogenetic protein 2(BMP-2) is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1(+) to construct hBMP-2 eukaryotic expression vector pCDNA3.1(+)-hBMP-2. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level recombinant human bone morphogenetic protein 2(rhBMP-2) was constructed by co-transfecting the expression vectors pCDNA3.1(+)-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.1 and 1 micromol/L. Western blot analyses showed a specific band of about 18 kD in reduced sample lane and a specific band of about 32 kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level (7.83 microg/24 h/10(6) cells) of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase(ALP) activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium(CM) for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.
Alkaline Phosphatase ; biosynthesis ; Animals ; Blotting, Western ; Bone Morphogenetic Protein 2 ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; CHO Cells ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Induction ; drug effects ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; Solubility