OBJECTIVESTo construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.

METHODSThe BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.

RESULTSPCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.

CONCLUSIONConstruction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; genetics ; Genetic Therapy ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Plasmids ; Polymerase Chain Reaction ; Tissue Engineering ; Transforming Growth Factor beta

OBJECTIVETo construct fluorescent eukaryotic cell expression vector with human bone morphogenetic protein-7 (hBMP-7) gene and to transfect mouse stromal cell line W-20-17 to detect the bioactivity of pEGFP-hBMP-7 in vitro.

METHODSpEGFP-hBMP-7 plasmid was constructed by subcloning technique and identified by enzyme cutting and electrophoresis. W-20-17 cells were transfected with pEGFP-hBMP-7 by means of lipofectamine-2000 media methods. Transfection efficiency and gene expression were evaluated by fluorescent microscopy. ALP, von Kossa and osteocalcin (OC) were tested to determined the phenotypes of osteoblast.

RESULTSAfter 48 hours, the gene transfection efficiency was 40%. Based on GFP and immunofluorescence of pEGFP-hBMP-7, there was the expression of aim gene. After gene transfection, there were not significant different of cell morphology feature and cell proliferation. ALP activity, the number of calcium nodules and the expression of OC increased.

CONCLUSIONSpEGFP-hBMP-7 with bioactivity was constructed, which could induce W-20-17 cells to differentiate to osteoblasts.

Animals ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Cell Line ; Genetic Vectors ; Humans ; Mice ; Osteogenesis ; Stromal Cells ; cytology ; Tissue Engineering ; Transfection

The seat of human intelligence is the human cerebral cortex, which is responsible for our exceptional cognitive abilities. Identifying principles that lead to the development of the large-sized human cerebral cortex will shed light on what makes the human brain and species so special. The remarkable increase in the number of human cortical pyramidal neurons and the size of the human cerebral cortex is mainly because human cortical radial glial cells, primary neural stem cells in the cortex, generate cortical pyramidal neurons for more than 130 days, whereas the same process takes only about 7 days in mice. The molecular mechanisms underlying this difference are largely unknown. Here, we found that bone morphogenic protein 7 (BMP7) is expressed by increasing the number of cortical radial glial cells during mammalian evolution (mouse, ferret, monkey, and human). BMP7 expression in cortical radial glial cells promotes neurogenesis, inhibits gliogenesis, and thereby increases the length of the neurogenic period, whereas Sonic Hedgehog (SHH) signaling promotes cortical gliogenesis. We demonstrate that BMP7 signaling and SHH signaling mutually inhibit each other through regulation of GLI3 repressor formation. We propose that BMP7 drives the evolutionary expansion of the mammalian cortex by increasing the length of the neurogenic period.
Animals ; Mice ; Humans ; Ependymoglial Cells/metabolism* ; Hedgehog Proteins/metabolism* ; Ferrets/metabolism* ; Cerebral Cortex ; Neurogenesis ; Mammals/metabolism* ; Neuroglia/metabolism* ; Bone Morphogenetic Protein 7/metabolism*

OBJECTIVEThis study explored the dynamic expression of the E3 ubiquitin-protein ligase gene, Arkadia, in response to carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and investigated the differential expression that occurs following treatment with the anti-fibrotic bone morphogenetic protein-7 (BMP-7).

METHODSThirty healthy male imprinting control region (ICR) mice were randomly assigned to three groups: normal (control; n = 6), CCl4-induced model group (model; n = 18), and CCl4-induced model with BMP-7 treatment group (treatment; n = 6). The model group was further divided into three subgroups (n = 6 each) for analysis at 4, 8 and 12 weeks after fibrosis induction. Liver fibrosis was induced by hypodermic injections of 60% CCl4 /peanut oil (5 mL/kg) to the hind legs of mice two-times per week in alternating legs for a period of 12 weeks. At week 9, the treatment group of CCl4-induced mice were given an intraperitoneal injection of BMP-7 (300 pg/g) simultaneously with that day's hypodermic injection of 60% CCl4 /peanut oil, and then every other day for a period of four weeks. The pathological changes in liver tissues were observed after staining with hematoxylin-eosin (HE) and Masson's trichrome. Messenger RNA (mRNA) and protein expression of Arkadia in liver were evaluated using reverse transcription-polymerase chain reaction and immunohistochemistry and Western blotting, respectively.

RESULTSMouse models of liver fibrosis were successfully established by CCl4 exposure. Arkadia, Smad7 and TGF-beta1 mRNA levels were up-regulated in the model group in a time-dependent manner (vs. control group), and BMP-7 treatment led to significant down-regulation of the CCl4-induced expression of the three genes (vs. control group: F = 812.80, 451.46, and 998.96, respectively; P less than 0.01). At week 12, the mRNA levels of Arkadia, Smad7, and TGF-b1 were significantly lower in the BMP-7 treatment group than in the model group (t = 12.108, 18.737, and 16.364, respectively; P less than 0.01). Arkadia, Smad7, and TGF-b1 protein staining was weak in the portal area of control liver tissue. In contrast, the model group showed significantly stronger staining for all three proteins in the portal area and in the cytoplasm of liver cells. The staining of Arkadia, Smad7, and TGF-b1 proteins was significantly lower in the treatment group (vs. control group: F = 8.399, 609.690, and 900.561, respectively; P < 0.01). At week 12, the protein levels of Arkadia, Smad7, and TGF-b1 were significantly lower in the treatment group than in the model group (t = 23.438, 11.667, and 42.889, respectively; P < 0.01).

CONCLUSIONArkadia expression gradually increased along with the development of liver fibrosis but was suppressed by treatment with the anti-fibrotic factor, BMP-7.

Animals ; Bone Morphogenetic Protein 7 ; pharmacology ; Liver ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred ICR ; Ubiquitin-Protein Ligases ; metabolism ; Up-Regulation

OBJECTIVETo explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs).

METHODSThe marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected.

RESULTSThe exogenous gene could be expressed efficiently in transduced BMSCs.

CONCLUSIONThe present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Cells, Cultured ; Genetic Vectors ; genetics ; Rabbits ; Recombinant Proteins ; biosynthesis ; Stromal Cells ; cytology ; metabolism ; Tibia ; cytology ; Transfection ; Transforming Growth Factor beta

OBJECTIVETo study the effect of recombinant pAd-BMP-7 on osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSC).

METHODSRecombinant pAd-bone morphogenetic protein (BMP) 7 was constructed and the titer of recombinant adenovirus was determined. pAd-BMP-7 and pAdTrack-CMV were used to transfect rat MSC. Transfection efficiency was measured by fluorescent microscope and BMP-7 expression was detected by RT-PCR and immunocytochemical analysis. The MSC were then randomly divided into 3 groups: group A received pAd-BMP-7 transfection, group B was transfected with pAdTrack-CMV, and group C received pAdTrack-CMV transfection plus bone supplements. Osteogenic differentiation of MSC was evaluated by examination of mineralization nodes formation.

RESULTSThe titer of pAd-BMP-7 reached about 2.0 x 10(15) pfu/L and transfection efficiency of exogenous gene was nearly 99% at day 2. The expression of exogenous gene sustained about 5 to 7 weeks, with a higher level during first 3 weeks. After transfection, transcription of BMP-7 and expression of BMP-7 protein were also verified in MSC. Compared with the negative results in group B, mineralization nodes were formed in both group A and group C. However, group A showed better formation of mineralization nodes than group C (P < 0.01).

CONCLUSIONSThe results of this study indicated that recombinant pAd-BMP-7 can successfully transfect rat MSC and accelerate their osteogenic differentiation. The technique explored in this study provides a unique and valuable gene engineering approach for reconstruction of craniofacial bone defects.

Adenoviridae ; genetics ; Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Cell Differentiation ; Cells, Cultured ; Genetic Vectors ; Mesenchymal Stromal Cells ; cytology ; Rats ; Transfection

In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3-rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilage.
Animals ; Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Cartilage, Articular ; metabolism ; Chondrocytes ; metabolism ; Gene Expression ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics ; metabolism

OBJECTIVETo examine the transfection of Homeobox A13 (HOXA13) on epithelial-mesenchymal transition (EMT) and the expression of bone morphogenetic protein-7 (BMP-7) induced by albumin-overload in human kidney tubular epithelial cells (HKCs).

METHODSThe cultured HKCs were treated with 20 mg/mL human serum albumin (HSA) for 48 hours. Protein expression of cytokeratin (CK), vimentin and HOXA13 in the HKCs was assessed by Western blot. Protein expression of CK, vimentin, and BMP-7 was also detected in HKCs transfected with lipofectamine contained HOXA13 DNA.

RESULTSHSA induced EMT in HKCs, presented by decreased CK expression (P<0.01) and increased vimentin expression (P<0.01). The up-regulated expression of HOXA13 transfected by lipofectamine inhibited the level of EMT induced by HSA in HKCs (P<0.05). The decreased rate of BMP-7 protein expression induced by HSA was inhibited by over-expressed HOXA13 in HKCs (P<0.05).

CONCLUSIONSTransfection of HOXA13 in HKCs could inhibit the degree of EMT induced by albumin-overload, possibly by increasing BMP-7 expression.

Bone Morphogenetic Protein 7 ; genetics ; Cells, Cultured ; Epithelial Cells ; metabolism ; Epithelial-Mesenchymal Transition ; Homeodomain Proteins ; physiology ; Humans ; Keratins ; genetics ; Kidney Tubules ; metabolism ; Transfection ; Vimentin ; genetics

OBJECTIVETo obtain highly efficient small interfering RNAs (siRNAs) against bone morphogenetic protein-7 (BMP-7) for future functional analysis of BMP-7 in bone homeostasis.

METHODSWe designed 4 independent siRNA sequences against rat BMP-7 and transfected them in rat umbilical vein endothelial cells. After 72 h, we examined BMP-7 mRNA and protein level in the transfected cells using real-time PCR and Western blotting, respectively.

RESULTSAll the 4 siRNAs effectively reduced BMP-7 expression in rat umbilical vein endothelial cells, and among them BMP-7-3 siRNA showed the highest efficiency.

CONCLUSIONWe have obtained an efficient siRNA to knockdown BMP-7 expression in vitro for further investigation of the role of BMP-7 in bone and cartilage formation.

Animals ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Cells, Cultured ; Endothelial Cells ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; Rats ; Real-Time Polymerase Chain Reaction ; Transfection

OBJECTIVETo investigate the effect of salvianolic-acid B (SA-B) on epithelia-mesenchymal transition in human renal proximal tubular cells (HK2), induced by transforming growth factor beta1 (TGF-beta1).

METHODEpithelia-mesenchymal transition (EMT) was induced with TGF-beta1 in HK2 cultured in vitro. Different concentrations (2, 5, 10, 20 microg x L(-1)) and stimulant periods (12, 24, 48 h) were tried to find the perfect condition for EMT. At the same time bone morphogenetic protein-7 (BMP-7, positive control) and the SA-B intervention were given to observe their effect on EMT. Western blot and immunofluorescent microscopy were used to analyze the expression of E-cadherin and alpha-smooth muscle actin (alpha-SMA) in HK2.

RESULTBMP-7 significantly inhibited the down-regulation of E-cadherin and the up-regulation of alpha-SMA induced by TGF-beta1 (P < 0.05), and SA-B significantly inhibited the up-regulation of alpha-SMA expression induced by TGF-beta1 (P < 0.05), but not the down-regulation of E-cadherin induced by TGF-beta1.

CONCLUSIONSA-B and BMP-7 can inhibit TGF-beta1-induced EMT in HK2. Their common role is to inhibit the up-regulation of alpha-SMA, and the effect of SA-B on the regulation of E-cadherin needs further study to be confirmed.

Benzofurans ; pharmacology ; Bone Morphogenetic Protein 7 ; pharmacology ; Cadherins ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Epithelial Cells ; pathology ; Humans ; Mesoderm ; pathology ; Transforming Growth Factor beta1 ; pharmacology