Osteogenic Potential of Cultured Bone Marrow Stromal Cells Transfected with Transforming Growth Factor β_1 Gene in vitro

Bone tissue engineering has a broad prospect intreating bone defects. It is a crucial link to chooseand optimize seed cells.In recent years,bone mar-row stromal cells(BMSCs) have been regarded to beone kind of preferred seed cells[1,2 ] . We have trans-fected BMSCs with TGF- β1gene and then examinedtheirosteogenic potential in orderto improve theirbi-ological properties.1　 MATERIALSAND METHODS1 .1　 Experimental MaterialsThe materials used in the experiment includedpc DNA3 - T…     

Study of Rat Osteoblasts Transfected by Transforming Growth Factor β1 Gene

Summary: In order to investigate the effect of TGFβ1 gene transfer on the biological characteristics,the effects of gene transfer and supernatant of transfected osteoblasts on the proliferation and ALP activity of osteoblasts were detected by 3H-TdR and MTT. Our results showed that TGFβ1 gene transfer had no effect on the biological characteristics and the activated supernatant of transfected os-teoblasts stimulated proliferation and inhibited ALP activity of osteoblasts. TGFβ1 gene transfer could promote the expression of TGFβ1 and the biological characteristics of transfected osteoblasts were sta-ble, which might be helpful for gene therapy of bone defects in vivo.     

Cloning and Expression of Rat Transforming Growth Factor β1 cDNA in Osteoblasts

In order to study transforming growth factor81 (TGF51) in molecular level and investigate thefeasibility of TGF51 gene therapy for bone defects,an eukaryotic expression vector containing ratTGF51 cDNA was constructed. The expression ofTGF51 in the transfected osteoblasts was observed.1 MATERIALS AND METHODS1. 1 Animals and Cell CultureSprague--Dawley rats, I adult and 5 newborn,were purchased from Animal Center of Tong nMedical University. Lymphocytes, isolated fromperipheral…     

Molecular Tissue Engineering: Applications for Modulation of Mesenchymal Stem Cells Proliferation by Transforming Growth Factor β_1 Gene Transfer 

It is well established that damaged articularcartilage has only a limited ability to healitself. Tissueengineering represents a new alternative approach tocartilage repairing. However,as it is very difficult toacquire seed cells with good activity,especially thoseinflammatory factors which induce the degradation ofarticular cartilage in the pathology of trauma orosteoarthritis were still exited,the success of all ofthese techniques has been variable and limited in thatnone were able to restore…     

Effect of Colquhoumia Root on the Expression of Transforming Growth Factor-β in Mesangial Proliferation Glomerulonephritis Model

ThechronicglomerulonephritisremainthemostcommondiseasetocausechronicrenalfailureinChina.Themesangialproliferationglomerulo nephritis(MsPGN)isthemostcommonpathologi caltypeoftheprimaryglomerulonephritis,ac countingforabout50%ofpatientsreceivingrenalpuncture[1].Reducingproteinuriaandmaintainingthekidneyfunctionisveryimportantforthetreat mentofthecondition.InthisstudyMsPGNmodelwasestablishedbyinjectionofanti thymocyteserum(ATS)inSDratsandproteinuria,pathologicalchangesofkid neyandTGFβ1exp…     

Influence of Exogenous TGFβ_1 on the Expression of Smad2 and Smad3 in Rat Bone Marrow-derived Mesenchymal Stem Cells

Bone marrow derived mesenchymal stem cells(MSCs) have multipotential to differentiate intoosteogenic, myogenic, and adipogenic cell linea ges, depending on signals derived from the cellularenvironment. At present, MSCs have been regar ded to be one kind of preferred seed cells in the re generation of bone defects. The TGFβ1 protein ismultifunctional peptide and has a broad range ofcellular activities including the control of cell pro liferation and differentiation …     

Expression of Transforming Growth Factor β1 in Mesenchymal Stem Cells: Potential Utility in Molecular Tissue Engineering for Osteochondral Repair

Summary: The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-β1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog. The transient and stable expression of TGF-β1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently trans fected MSCs in vitro with the TGF-β1 gene causing a marked up-regulation in TGF-β1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were sus ceptible to in vitro lipofectamine mediated TGF-β1 gene transfer and that transgene expression persist-ed for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, I.e.molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineer-ing, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness artic ular cartilage defects that occur as a result of injury and osteoarthritis.     

Effect of Antisense MBD1 Gene Eukaryotic Expression Plasmid on Expression of MBD1 Gene in Human Biliary Tract Carcinoma Cells

DNAmethylationhasavarietyofimportantfunctionsintheregulationofgeneexpression[1].ManystudiesdemonstratedthatthepathogenesisofbiliarytractcarcinomawascloselyassociatedwiththeaberrantDNAmethylationstatusoftumorrelatedgenes.TheaberrantstatusofDNAmethylationincludedthehypomethylationofonco genesandthehypermethylationoftumorsuppres sorgenes(TSG).IncreasingtheexpressionofTSGandimprovingthefunctionofTSGbyelimi natingitshypermethylationstatusmaybeanewwaytotreatbiliarytractcarcinoma.Toexplorether…     

Effects of Heparin on Transforming Growth Factor-β_1 and Extracellular Matrix Components in the Glomeruli of Diabetic Rats

The key mechanism of diabetic glomerular scle-rosis is proliferation of mesangial cells and overaccu-mulation of extraceluar matrix(ECM) .Research onthis mechanism has been done in recent years,butreports on the relationship between growth factorandexpression of ECM,especially ordinary heparin' sregulation on this expression,are not enough in doc-uments. In orderto understand betterthe mechanismof diabetic nephropathy and prospect the clinical ap-plication of ordinary heparin' s preventive a…     

Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

Arresten,human noncollagenous domain 1 oftheα1 chain of type Ⅳcollagen,is derived fromthe carboxy terminal of type Ⅳcollagen[1].It wasidentified as a powerful inhibitor of angiogenesisrecently .In earlier studies , human arresten wassuccessfully amplified from placenta tissue , andmolecular cloning and DNA sequencing verifiedthat the coding sequence obtained was correct[2].Prokaryotic expression vector of arresten gene hasbeen constructed[3]. Excessive proliferation andmigration to endome…     

Effect of IL-1βand TNF-αon the Expression of MonocyteChem otactic Protein-1 in Endom etriotic Cells

Ｅｎｄｏｍｅｔｒｉｏｓｉｓｉｓａｍｏｎｇｔｈｅｍｏｓｔｃｏｍ－ｍｏｎｇｙｎｅｃｏｌｏｇｉｃｅｎｔｉｔｉｅｓ，ｃｈａｒａｃｔｅｒｉｚｅｄｂｙｅｃｔｏｐｉｃｇｒｏｗｔｈｏｆｅｎｄｏｔｈｅｌｉａｌｔｉｓｓｕｅｉｎｔｈｅｐｅｒｉｔｏｎｅａｌｃａｖｉｔｙ．Ａｌｔｈｏｕｇｈｔｈｅｐａｔｈｏｇｅｎｅ－ｓｉｓｉｓｓｔｉｌｌｐｏｏｒｌｙｕｎｄｅｒｓｔｏｏｄ，ａｃｏｎｓｉｓｔｅｎｔｆｉｎｄｉｎｇｉｎｔｈｅｐｅｒｉｔｏｎｅａｌｆｌｕｉｄｏｆｔｈｅｓｅｐａ－ｔｉｅｎｔｓｈａｓｂｅｅｎｔｈｅｉｎｃｒｅａｓｅｄｎｕｍｂｅｒ…     

Modulation of Matrix Metalloproteinase and TIMP-1 Expression by TGF-β1 in Cultured Human RPE Cells

Matrix metalloproteinases(MMPs)are a familyof zinc binding,calcium-dependent endopeptidases thatfunction by degrading extracellular matrix(ECM)components.The functional effects of these enzymesare in part controlled byinteractions withtissueinhibi-tors of metalloproteinases(TI MPs)acting as naturalMMPinhibitors.Aprecise balance between MMPandTI MPactivities may be i mportant for the integrity ofECMcomponents[1].It shows that the expression andactivity of MMPs could be regulated by va…     

Effects of Mycophenolic Acid on High Glucose-induced Expression of TGF-β and CTGF in Mesangial Cells

Mycophenolicacid(MPA),theactivemetab oliteofmycophenolatemofetil(MMF),isapo tent,non competitive,andreversibleinhibitorofinosine5'monophosphatedehydrogenase(IMP DH),therate limitingenzymefordenovopurinesynthesis,andthusinterfereswithcellularbiosyn thesisofguanosineanddeoxyguanosinenucleo tides.BesidesstronglyinhibitingtheproliferationofTlymphocyteandBlymphocyte,MPAhasbeenidentifiedtohavesomeotherbiologicalfunctionsinrecentyears,includinginterferingglycosylationofadhesionmolecule,relievin…     

Transforming Growth Factor-β_2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

Primary open- angle glaucoma (POAG) is aleading cause of blindness,which involves optic neu-ropathy accompanied by characteristic visual field de-fects and is often associated with elevated intraocularpressure due to disturbance ofaqueoushumor outflowthrough the trabecularmeshwork (TM) .The patho-physiology of the TM in POAG has been character-ized by an increasein extracellularmatrix componentsand a decrease in the number of TM cells[1,2 ] .It hasbeen found that,compared with the non- P…     

Binding of EGF1 Domain Peptide in Coagulation Factor Ⅶ with Tissue Factor and Its Implications for the Triggering of Coagulation

The binding function of EGF1 domain peptide with tissue factor(TF)and its ability of triggering coagulation were explored.The TF expression model in vitro was established by lipopolysaccha-ride induction.The affinity of EGFP-EGF1 and TF expressing cells was analyzed by fluorescence microscopy and flow cytometry(FCM).The affinity of EGFP-EGF1 and rat soluble TF was quantitated by surface plasmon resonance(SPR).The ability of EGFP-EGF1 in triggering coagulation was tested by prothrombin time assay.The FCM res...     

Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

Background  Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.
Methods  Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.
Results  The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.
Conclusion  Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

     

Inhibitory Effect of TanshinoneⅡA on TGF-β1-induced Cardiac Fibrosis

This study examined the effect of tanshinoneⅡA (TSNⅡA) on the cardiac fibrosis induced by transforming growth factor β1 (TGF-β1) and the possible mechanisms. Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley (SD) rats by the trypsin digestion and differential adhesion method. The cells were treated with 5 ng/mL TGF-β1 alone or pretreated with TSNⅡA at different concentrations (10–5 mol/L, 10–4 mol/L). Immunocytochemistry was used for cell identification, RT-PCR for detection of the mRNA expression of connective tissue growth factor (CTGF) and collagen type Ⅰ (COLⅠ), Western blotting for detection of the protein expression of Smad7 and Smad3, and immunohistochemistry and immunofluorescence staining for detection of the protein expression of phosphorylated Smad3 (p-Smad3), CTGF and COLⅠ. The results showed that TGF-β1 induced the expression of CTGF, COLⅠ, p-Smad3 and Smad7 in a time-dependent manner. The mRNA expression of CTGF and COLⅠ was significantly increased 24 h after TGF-β1 stimulation (P<0.01 for all). The protein expression of p-Smad3 and Smad7 reached a peak 1 h after TGF-β1 stimulation, much higher than the baseline level (P<0.01 for all). Pretreatment with high concentration of TSNⅡA resulted in a decrease in the expression of p-Smad3, CTGF and COLⅠ (P<0.01). The protein expression of Smad7 was substantially upregulated after pretreatment with two concentrations of TSNⅡA as compared with that at 2h post TGF-β1 stimulation (P<0.05 for low concentration of TSNⅡA; P<0.01 for high concentration of TSNⅡA). It was concluded that TSNⅡA may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the TGF-β1-induced phosphorylation of Smad3 and partially blocking the TGF-β1-Smads signaling pathway.     

Connective Tissue Growth Factor Expression in Rabbit Corneal Fibroblast and Its Effect on Fibroblast Proliferation In Vitro

Connective tissue growth factor (CTGF) takespart in chemotactic,mitogenic,cell matrix inducingevents and scar formation.CTGF can activate andregulate the process that concerns with wound repairand regeneration.Itcan promote fibroblastprolifera-tion and extracellular matrix synthesis and secretion,stimulate vascular endothelial cell regeneration andneovascularization.CTGF is involved in many patho-genesis,such as dermatosclerosis,breasttumor infil-tration and metastasis,proliferating nephri…     

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

Hepatocellularcarcinoma (HCC)isamajorcauseofcancerdeathwithmorethan 1.2millionglobalan nualincidences[1] .Surgeryandlivertransplantationaretheonlyeffectivetreatments ,butmostHCCpa tientsarenoteligiblebecauseofdiagnosisatalaterstageorunderliverinsufficiencyinthesettingofcir rhosis[2 5] .Thedevelopmentofnoveltreatmentstrategiesisneeded .TheAFPisanHCC associatedoncofetalantigen .ThemajorityofhumanHCCsoverexpresstheoncofetalantigenAFP .ThisMr 700 0 0 glycoprotein ,producedathighlevelsbyth…