OBJECTIVETo acquire sufficient PDGF-BB protein and provide the basis for the further studies of its role on the fracture healing and trauma reconstruction and its clinical applications.

METHODSConstructed the prokaryotic expression vector pQE-PDGF-B with the gene rearrangement technique, and the monomeric form of recombinant PDGF-B expressed in E. coli M15.

RESULTSPDGF-B mature peptide gene was inserted into prokaryotic expression vector pQE30, which was confirmed by PCR, enzyme digestion and sequencing identification; the expressed products of pQE-PDGF-B in E. coli showed a single protein on SDS-PAGE, and their expression level was about 15% of the total bacterial protein. The molecular weight of the purified PDGF-B protein was about 15 KDs on SDS-PAGE.

CONCLUSIONSThe construction of recombinant plasmid and preparation of the monomeric protein of PDGF-B provides a solid foundation for further studying the function of PDGF-BB and producing biologically PDGF-BB protein.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; In Vitro Techniques ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-sis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the role of spleen tyrosine kinase (syk) in the phenotypic modulation induced by platelet-derived growth factor (PDGF-BB) in rat pulmonary vascular smooth muscle cells (VSMC).

METHODSVascular smooth muscles were isolated from pulmonary media of SD rats, cultured, adopted, and divided into 3 groups: blank control group, control group and medicine intervention group. The changes of proliferation and ultrastructure of vascular smooth muscle cells by using [(3)H] thymidine incorporation and electron microscopy. The mRNA and protein expression level of syk, alpha-smooth muscle-actin (α-SM-actin) and smooth muscle protein 22alpha (SM22α) were detected by RT-PCR and Western blotting. The change of fluorescence intensity was detected by laser scanning confocal microscope.

RESULTSTreatment with PDGF-BB for 24 h resulted in a significant increase in [(3)H] thymidine incorporation (2429.25 ± 253.36 vs. 242.75 ± 14.33,P < 0.01) and marked change in phenotype and cytoskeleton, the level of average optical density decreased significantly (263.75 ± 19.21 vs.1146.23 ± 62.61, P < 0.01). Meanwhile, the mRNA (1.70 ± 0.25 vs. 1.01 ± 0.12, P < 0.05) and protein level of syk significantly increased, the mRNA and protein expression of α-SM-actin (0.10 ± 0.00 vs. 1.00 ± 0.00, P < 0.01) and SM22α (0.18 ± 0.00 vs. 1.00 ± 0.01, P < 0.01) significantly decreased in VSMC induced by PDGF-BB. Piceatannol could inhibit significantly these biological effects. Compared with control group, the level of [(3)H] thymidine incorporation (527.00 ± 27.76 vs. 2429.25 ± 253.36,P < 0.01) was significantly down-regulated and the VSMC presented an apoptotic status in medicine intervention group, the level of average optical density increased significantly (810.65 ± 37.94 vs. 263.75 ± 19.21,P < 0.01) in medicine intervention group. Meanwhile, the mRNA (0.36 ± 0.07 vs. 1.70 ± 0.25, P < 0.01) and protein level of syk significantly decreased. The mRNA and protein levels of α-SM-actin (0.22 ± 0.00 vs. 0.10 ± 0.00, P < 0.01) and SM22α (0.31 ± 0.00 vs. 0.18 ± 0.00, P < 0.01) were significantly higher in medicine intervention group than in control group. The level of average optical density increased significantly (810.65 ± 37.94 vs. 263.75 ± 19.21, P < 0.01).

CONCLUSIONSyk plays an important role in vascular remodeling by changing the phenotypes and cytoskeleton of VSMC stimulated by PDGF-BB.

Animals ; Cells, Cultured ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Phenotype ; Platelet-Derived Growth Factor ; genetics ; Protein-Tyrosine Kinases ; genetics ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Sprague-Dawley ; Syk Kinase

OBJECTIVETo study the role of platelet-derived growth factor (PDGF) in the pathogenesis of preeclampsia (PRE).

METHODSThirteen normal and 20 PRE late-pregnancy women were enrolled in this study. The serum PDGF-BB levels were measured with enzyme-linked immunosorbent assay, and the expression of PDGF-B mRNA in the decidual blood vessel was determined using in situ hybridization.

RESULTSPDGF-BB levels in PRE group was significantly higher than that in normal pregnant women (83.54 -/+34.52 vs 39.61-/+18.20, P<0.001), and the expression of PDGF-B mRNA in decidual blood vessel was also significantly higher in PRE group (P<0.001), showing a positive correlation between serum PDGF and PDGF-B mRNA expression (r=0.603, P<0.001).

CONCLUSIONPDGF is associated with the pathology of decidual blood vessel. Elevated serum PDGF levels and PDGF-B mRNA expression in the decidual blood vessel may play an important role in the pathogenesis of PRE.

Adult ; Decidua ; blood supply ; Female ; Gene Expression Regulation ; Humans ; Platelet-Derived Growth Factor ; genetics ; metabolism ; Pre-Eclampsia ; blood ; genetics ; metabolism ; pathology ; Pregnancy ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo express human platelet-derived growth factor (hPDGF) B chain mature peptide gene in a prokaryotic expression system and detect the bioactivity of the expressed protein.

METHODShPDGF B chain mature peptide gene was amplified and expressed in E. coli, and the recombinant protein, rhPDGF-BB, was purified and renatured in GSSG/GSS system. The bioactivity of rhPDGF-BB in vitro was evaluated with SD rat osteoblasts.

RESULTSThe full-length PDGF-B mature peptide gene was obtained and verified, and successfully expressed in E. coli. Bioactivity detection results showed that the expressed rhPDGF-BB obviously promoted the proliferation and DNA replication of SD rat osteoblasts in vitro (P<0.01).

CONCLUSIONhe PDGF-B chain mature peptide cDNA has been successfully cloned and the PDGF-B precursor highly expressed in E. coli, and renatured rhPDGF-BB displays high bioactivity as shown by MTT assay and flow cytometry. This success provides the basis for production of functional PDGF-BB and facilitates further studies of its role in fracture healing and trauma reconstruction.

Animals ; Cell Proliferation ; Cells, Cultured ; DNA Replication ; Escherichia coli ; genetics ; Genetic Vectors ; Humans ; Osteoblasts ; metabolism ; Proto-Oncogene Proteins c-sis ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo detect whether the activation of nuclear factor-kappa B (NF-kappaB) in endothelial cells induced by mm-LDL can promote platelet-derived growth factor-B (PDGF-B) expression in vitro, and whether it is also present in hypercholesterolemic rats in vivo, influence of age on NF-kappaB and PDGF-B signal transduction pathway.

METHODSEstablished hypercholesterolemic rat model by feeding with a high-cholesterol ration. The activation of NF-kappaB in aortic endothelial cells was identified by immunohistochemical staining, the expression of PDGF-B mRNA and PDGF-B protein were examined using in situ hybridization and immunohistochemistry respectively.

RESULTSIn comparison with the control rats, a positive immunostaining of NF-kappaB in nuclei of aortic endothelial cells of the experimental rats was detected after a high cholesterol ration for 6 weeks. The number of endothelial cells expressing PDGF-B mRNA increased and the intensity was dependent upon the duration of high-cholesterol intake. NF-kappaB translocation (0.461 +/- 0.075 vs. 0.350 +/- 0.094, P < 0.05) and PDGF-B expression in 10-month old Wistar rats were more remarkable than that of 2-month old rats after having cholesterol for 16 weeks. Immunohistochemical staining for PDGF-B gave a similar result (0.230 +/- 0.040 vs. 0.185 +/- 0.037, P < 0.001).

CONCLUSIONSHypercholesterolemia is capable of activating nuclear translocation of NF-kappaB and promoting expression of PDGF-B in rat aortic endothelial cells in vivo, this coincided with the results obtained in ox-LDL or mm-LDL experiments on endothelial cells in vitro. This phenomenon is much more evident in 10-month old rats which indicates that age might have a close relationship with NF-kappaB - PDGF-B signal transduction pathway.

Active Transport, Cell Nucleus ; Age Factors ; Animals ; Aorta ; metabolism ; Arteriosclerosis ; etiology ; Endothelial Cells ; metabolism ; Hypercholesterolemia ; metabolism ; Immunohistochemistry ; Male ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins c-sis ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

OBJECTIVETo study the effects of Jiawei Huzhang San (JWHZS) decoction on the expressions of the inflammatory factors monocyte chemoattractant protein-1 (MCP-1) and platelet-derived growth factor-BB (PDGF-BB) on experimental autoimmune prostatitis in rats.

METHODSTwelve male Wistar rats were taken as normal controls, and models of experimental autoimmune prostatitis were established in another 60 by injection of SC purified prostate protein with FCA, and then divided into five groups to be treated with normal saline, indomethacin, high-dose JWHZS (0.445 g/kg), medium-dose JWHZS (0.223 g/kg) and low-dose JWHZS (0.089 g/kg), respectively. All the rats were sacrificed at 30 days after the treatment for detection of the mRNA and protein expressions of inflammatory factors by immunohistochemistry and fluorescent quantitative RT-PCR.

RESULTSIn the high-, medium- and low-dose JWHZS groups, the mRNA expressions of MCP-1 (0.31 +/- 0.14, 0.49 +/- 0.21 and 0.62 +/- 0.28) and PDGF-BB (0.50 +/- 0.22, 0.54 +/- 0.17 and 0.71 +/- 0.29), and the protein expressions of MCP-1 (677 +/- 208, 725 +/- 311 and 1302 +/- 884) and PDGF-BB (1265 +/- 698, 1347 +/- 827 and 1655 +/- 812) were significantly lower than in the model control group (MCP-1 mRNA: 1.12 +/- 0.43; MCP-1 protein: 2201 +/- 934; PDGF-BB mRNA: 1.14 +/- 0.51; PDGF-BB protein: 2754 +/- 852) (P < 0.05). And JWHZS exhibited a significantly better activity at high and medium doses than at a low dose (P < 0.05). In the indomethacin control group, both the mRNA and protein expressions of MCP-1 (0.71 +/- 0.34 and 1824 +/- 1157) and PDGF-BB (1.08 +/- 0.37 and 2493 +/- 924) were markedly higher than in the JWHZS groups (P < 0.01).

CONCLUSIONDown-regulation of the inflammatory factors MCP-1 and PDGF-BB may be the important molecular mechanism of JWHZS acting on experimental autoimmune prostatitis.

Animals ; Autoimmune Diseases ; drug therapy ; metabolism ; Chemokine CCL2 ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; therapeutic use ; Inflammation ; Male ; Phytotherapy ; Platelet-Derived Growth Factor ; metabolism ; Prostatitis ; drug therapy ; metabolism ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

Cell Line ; Epidermal Growth Factor ; biosynthesis ; genetics ; Fibroblast Growth Factor 2 ; biosynthesis ; genetics ; Hepatocytes ; metabolism ; Humans ; Interleukin-10 ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVEIt is known that intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in liver fibrogenesis. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and on the platelet-derived growth factor-B (PDGF-B).

METHODSIn vitro, hepatic stellate cell (HSC)-T6 cell line was treated with Aldo for 10 min, 0.5 h, 1 h, 2 h and 3 h. Protein expression of phospho-p42/44 was detected by Western blot. In addition, HSC-T6 were preincubated for 1 h or not at all with U0126 (an inhibitor of the MAPK/ERK kinase), and antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expressions of phospho-p42/44 and PDGF-B were measured by Western blot. DNA biding activity of EGR-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of immunohistochemistry, expression of PDGF-B was detected.

RESULTSAldo induced phospho-p42/44 expression could be abrogated by U0126; NAC did not inhibit phospho-p42/44 expression. Gel shift study showed that stimulation of HSC by Aldo markedly increased the EGR-1 DNA binding activity, which was abrogated by U0126, reaching a maximum at 60 minutes, and then declined progressively. NAC did not reduce the EGR-1 activity. Aldo increased the PDGF-B protein level in HSC, which was not attenuated by NAC and U0126.

CONCLUSIONSStimulation of HSC by Aldo results in activation of EGR-1 via ERK1/2 pathway, leading to up-regulation of PDGF-B expression.

Aldosterone ; pharmacology ; Cell Line ; Early Growth Response Protein 1 ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-sis ; biosynthesis ; genetics ; Signal Transduction

OBJECTIVETo study the effect of platelet derived growth factor-B and its receptor expression on the proliferation of renal cell carcinoma ACHN cells in vitro and in vivo.

METHODSPDGF-B gene was transfected into human renal carcinoma cell line ACHN cells, and the proliferation capability of ACHN cells transfected with or without PDGF-B was assessed by MTT assay. The effect of PDGF-B on the expression of p-PDGFR-β in endothelial cells and vascular smooth muscle cells (VSMC) was detected by Western blot. ACHN cells transfected with PDGF-B were injected into mice (untransfected ACHN as control) to induce tumor formation. Immunohistochemical staining was used to detect the expression of Ki-67 in tumor cells and the tumor volume was measured to compare the tumor growth in the two groups.

RESULTSThe PDGF-B expression of ACHN cells in transfected group was significantly increased than that in the untransfected group. MTT assay showed that the proliferation capability of ACHN cells in the transfected and untransfected groups had no significant differences at different time points (P>0.05). The expression of p-PDGFR-β in VSMC was significantly increased when cultured with PDGF-B overexpression culture medium. The mean tumor size of the PDGF-B group and control group was (0.305±0.108) cm(3) and (0.577±0.218) cm(3), respectively (P=0.007). Ki-67-positive tumor cells were (41.00±5.34)/HPF in the PDGF-B-transfected group and (55.80±2.95)/HPF in the untransfected group (P=0.001).

CONCLUSIONPDGF-B overexpression may up-regulate p-PDGFR-β expression of VSMC in renal cell carcinoma, and inhibit the tumor cell proliferation and tumor growth through paracrine signaling.

Animals ; Carcinoma, Renal Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; Mice ; Proto-Oncogene Proteins c-sis ; Receptor, Platelet-Derived Growth Factor beta ; genetics ; metabolism

AIMTo observe expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC during early embryonic vascular development, and to initially investigate the differentiation effect of platelet derived growth factor-BB (PDGF-BB) on vascular smooth muscle cells (VSMCs) during that period.

METHODSMurine embryonic stem cell line expressing the enhanced green fluorescent protein (GFP) under the transcriptional control of the smooth-muscle-specific SM22alpha promoter was used to make embryoid bodies,and to analyze the expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC by immunofluorescence stainings, RT-PCR and Western blot. Then AG1296 (PDGF receptor inhibitor) 0 micro-mol/L(control group), 10 micromol/L and 50 micromol/L were used to treat EBs respectively in order to analyze the differences of SMa-actin, SM22alpha, myocardin and SMMHC at gene and protein levels among the three groups.

RESULTSSMalpha-actin, myocardin, SM22alpha and SMMHC expression in EBs were found to begin at day 0 (ESCs), 8, 11, 13 respectively during early embryonic vascular development. There were no clear differences in SMa-actin, SM22alpha, myocardin and SMMHC protein expression and SM22alpha, myocardin and SMMHC mRNA level among the three groups of different concentrations of AG1296.

CONCLUSIONA spontaneous VSMCs differentiation occurs during EBs development, SMalpha-actin is the first to be detected,the following are myocardin, SM22a and SMMHC. PDGF-BB may not be indispensable for the regulation of expression of VSMCs markers during early EBs differentiation.

Actins ; genetics ; metabolism ; Animals ; Biomarkers ; metabolism ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; metabolism ; Mice ; Microfilament Proteins ; genetics ; metabolism ; Muscle Proteins ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Nuclear Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-sis ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism