| Background and ObjectiveTissue engineering heart valve(TEHV) includes three main aspects:seeding cells, matrix material,valves forming and regenerating.The first step of the construction of TEHV in vitro is to seed cells on the scaffold.Firstly,the cells must adhere on the scaffold and then they can immigrate,differentiate and proliferate.The cell-adhesion on scaffold is the mainstay in these biological processes.Generally,the scaffolds are made of two kinds of biomaterials:artificial synthesized material and natural material,but they can not provide appropriate interface to the cells to attach and grow.Moreover,with TEHV development the researchers have discovered that seeding cells cultured in vitro proliferated slowly and adhered poorly.Simon has recently found decellular porcine aortic valves still had immunogenicity,especially in pediatric patients.So how to promote the seeding cells to adhere on scaffold is a key in the study of TEHV.Transforming growth factor-β1(TGF-β1) is a high-activity,multi-functional biological signaling molecule.Coding TGF-β1 genes lies in 19,q13 in the human genome.TGF-β1 exists in most mammals and is highly homologous.The biological roles of TGF-β1 involve inhibiting some kinds of cells proliferation,promoting certain mesenchymal cells proliferation,promoting extracellular matrix synthesis,inhibitition of matrix degradation and inducing cell differentiation.In the first step of our study,we constructed recombinant adenovirus coding for TGF-β1 gene.Secondly,we transfected them into BMSCs after we got sufficient BMSCs. To evaluate the effect of transfecting Ad.TGF-β1 into BMSCs,trancfected BMSCs were seeded onto the decellularized matrices so as to study their adhering and growing characteristics.Methods1.Construction of recombinant adenovirus coding for TGF-β1 geneTotal RNA was isolated from human spleen tissue using standard procedures.RT-PCR was performed to synthesize and amplify the TGF-β1 DNA which was further purified using electrophoresis and cleaved with appropriate restriction enzymes.The DNA was inserted into plasmid pET28.Human embroynic kidney 293 cells were transfected with DNA-TPC and recombinant adenovirus cosmid vector pAxcAwt.TGF-β1 by the positive lipofectamine method.Virus clones were isolated and propagated for restriction analysis. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated in 293 cells.Cytopathic effects(CPEs) of adenovirus were evaluated on Hela cells.2.Isolation,culture of human BMSCs and expression of TGF-β1 after transfection(1)Bone marrow was aspirated from iliac crest.BMSCs were obtained from isolation of the aspirate with Percoll step gradient,cultured and expanded in DMEM medium, characterized by flow cytometry analysis of the expression of CD34,CD71,CD44 and Desmin and immunohistochemical staining of them.(2)Transfection efficiency of adenovirus vector to BMSCs were identified by infection with various titrations of Ad.GFP.After Ad.TGF-β1 transfection,TGF-β1 protein expression in BMSCs was measured by methods of immunohistochemical staining,RTPCR respectively and the proliferation and growth of BMSCs were studied with cell counting and MTT assay.3.To study enhancing the adhesion of transfecting Ad.TGF-β1 into BMSCs(1)The porcine aortic valve leaflets were decellularized with the protocols:0.025% trypsin(8 hours)+1%DCA+ nuclease(24 hours),with continuous shaking at 37℃.Cell extraction efficiency was assessed by histological examination of decellularized matrices with HE staining and electron microscopy.The decellular valve leaflets were divided into three groups by seeding three different kinds of BMSCs(control group,transfected Ad.GFP group,transfected Ad.TGF-β1 group).The valve leaflets(n=8) were used as scaffold and BMSCs were seeded onto them three times at 24h interval.Seven days later,the adhesion and growth of BMSCs on the TEHV leaflets were studied with cell counting,MTT,HE staining,electron microscopy and characterized by flow cytometry analysis of the expression of CD34 and factorⅧ.The contents of valve collagen were judged by detecting valve hydroxyproline.(2)The valve leaflets were divided into three groups(divided as above).After the valve leaflets were placed into the flow field for 24 hours,the adhesion and growth of BMSCs on the TEHVs were assessed in same ways.Results1.Recombinant Ad vectors were constructed sucessfully.Transfection of TGF-β1 gene to BMSCs by adenovirus was safe and highly efficient. 2.In primary culture BMSCs attached and spread in triangular,polygonal or spindle-shaped morphology.Colonies were formed after 3-5 days.In passaged culture BMSCs became uniformly spindle-shaped,and expanded exponentially.Immunohistochemicalstaining and flow cytometry analysis indicated that BMSCs were positive for CD71,CD44 and negtive for CD34 and Desmin.BMSCs were effectively transfected by Ad.GFP in vitro. Over 95%of BMSCs were transfected at MOI 50.Human TGF-β1 protein was detected by immunohistochemical staining and RT-PCR result showed that the TGF-β1 transcription were identified in BMSCs 72 hours after gene transfer.3.Using the decellularization with the protocols:0.025%trypsin(8 hours)+1%DCA+ nuclease(24 hours),native cells in porcine aortic valve leaflets,at the same time the fibrous matrix retained in good condition.Cell adhesion assay:The MTT absorbency value(D570) of TGF-β1 group was more than control group,Ad.GFP group,there was no significant difference between control group and transfected Ad.GFP group.Histological analysis showed that the surface of TEHV leaflets of Ad.TGF-β1 group was covered with multilayered cells and the cells arrayed tightly,when seeded with BMSCs.There were no complete cells layer on the leaflets of control group and Ad.GFP group,cells arrayed out of order and somewhere the scaffold was naked.SEM demonstrated that the surface of TEHV leaflets of Ad.TGF-β1 group was smooth and confluent,matrix sediment was observed between the cells.The cells of control group and Ad.GFP group arrayed out of order and somewhere the scaffold was naked.Cells counting and MTT assay showed there were cells Ad.TGF-β1 group than of control group and Ad.GFP group.Moreover,the content of hydroxyproline of Ad.TGF-β1 group was more than that of control group and Ad.GFP group.After constructed TEHV leaflets in vitro and underwent the flow for 24 hours.Cells counting and MTT assay showed that BMSCs on the leaflets of control group and Ad.GFP group had residue about 50%.While those leaflets of Ad.TGF-β1 group had residue about 70%.Meanwhile,the morphological changes of residual cells were found by flow cytometry analysis.Ad.TGF-β1 group had more cells density than TGF-β1 group and cells adhered more tightly.Conclusions:1.The efficient and reliable recombinant adenovirus coding for TGF-β1 gene are constructed successfully.Transduction of TGF-β1 gene to BMSCs by adenovirus is highly efficient and safe.2.BMSCs are ideal for seeding cells and have extensive potential of proliferation. They can be obtained from bone marrow aspiration which cause only little trauma to the body.BMSCs can be infected with Ad.TGF-β1 efficiently in vitro and TGF-β1 protein is successfully produced.3.Transfceting TGF-β1 can promote adhesion,proliferation and extracellular matrix synthesis of BMSCs on decellular aortic valves.
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