Construction Of Co-expression Of HTK1and HTIMP1Genes Mediated By Recombinant Adenovirus Vector And Its Effects On Proliferation Of Rat Vascular Smooth Muscle Cells

Objectives Over-expression of human tissue kallikrein1(hTK1) gene has previously beenshown to partially inhibit the proliferation of arterial smooth muscle cells (VSMCs) both in ratvascular restenosis models and in vitro. However, the synergistic effects of combination withhTK1and tissue inhibitor of matrix metalloproteinase (TIMP) on improving vascular restenosisremains to be determined. In present study, we firstly connstructed co-expression of hTK1and hTIMP1gene mediated by recombinant adenoviral vector (Co-expression vector) andobserved its infection rate and expression. Then we investigated the inhibition protective roleof co-expression vector on the proliferation of VSMCs induced by platelet-derived growthfactor (PDGF-BB).MethodsPart I1Bought original plasmid containingh hTIMP1gene from Invitrogen company to constructedthe pDC316-hTIMP1-EGFP plasmid carrying enhanced green fluorescent protein(EGFP),then identified by polymerase chain reaction(PCR), enzyme digestion andsequencing analysis.2The CMV-hTIMP1fragment was amplified and recycled from the pDC316-hTIMP1-EGFPplasmid digested by the restriction endonuclease Bag Ⅲ/Sa Ⅱ.Meanwhile,digested thepDC316-hTK1vector (saved in laboratory) with the same restriction endonucleas.Transfectedthe E. Coli DH-5ɑ with the above products after recycled and connected by the Method ofheat shock,then selected the right targets from them to clone. Constructed a dual-geneco-expression shuttle plasmid pDC316-cmv-hTK1-cmv-hTIMP1,which was identified by polymerase chain reaction(PCR), enzyme digestion and sequencing analysis.3The linearized pDC316-hTIMP1-EGFP and pDC316-cmv-hTK1-cmv-hTIMP1，togetherwith the plasmid pBHGloxE1,3Cre were co-transfected into the293A cells,to package aco-expression recombinant adenoviral vector,Identified the target gene of the viruses by themethod of PCR.Detected the concentration by TICD50.PartII：1Primaty culture the thoracic aorta VSMCs of Sprague-Dawley rat in vitro pattern,andidentified VSMCs by fluorescent immunohistochemistry with Alpha Smooth Muscle Actinmonoclonal antibody.2Detect the infection rate by fluorescence microscopy and flow cytometry.3The VSMCs proliferation induced by PDGF-BB was accessed by cell counting and methylthiazolyl tetrazaliuin(MTT).ResultsPartI:1The shuttle plasmid pDC316-hTIMP1-EGFP and pDC316-cmv-hTK1-cmv-hTIMP1weresuccessfully constructed and identified by PCR, enzyme digestion and sequencing analysis,2The funtional Ad5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1were packed in293A cells by thethe shuttle plasmid pDC316-hTIMP1-EGFP and pDC316-cmv-hTK1-cmv-hTIMP1togetherwith the backbone plasmid pBHGloxE1,3Cre, together with the backbone plasmidpBHGloxE1,3Cre transfected into293packaging cells,successfully amplified and concentratedinto funtional Ad5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1. and identified the target gene ofthe viruses by the method of PCR3The titers of virus determined by TICD50method: Ad5-hTIMP1-EGFP为7.0×1011vp/ml，Ad5-hTK1-hTIMP1为8.5×1011vp/ml。PartII：1The primary VSMCs were successfully cultured, which contained red filamentous stripe inthe cytoplasm is identified to be positive. the purity is more than95%2The expression of EGFP in VSMCs reached up to99.57%by fluorensce microscope and flow cytometry after VSMCs were transfected Ad5-hTK1-IRES-EGFP in MOI-dependent(20-100MOI) and time-dependent (1-4d) manner.The expression of EGFP in VSMCs reachedup to98.14%after VSMCs were transfected Ad5-hTIMP1-EGFP in MOI-dependent(20-150MOI) and time-dependent (1-4d) manner.3The Cell counting and MTT method results showed that PDGF-BB can significantlypromoting cell proliferation and the control virus had no effect in cellproliferation..Proliferation of VSMCs induced by PDGF-BB were inhibited after thetransfection of the three kinds recombinant adenovirus in MOI-dependent (20-150MOI) andtime-dependent (2-5d) manner.Compared with the single-gene-exprssing vectors,Ad5-hTK1-hTIMP1significantly enhanced the inhibitory effect on cell proliferation(P<0.01).In the forth day,when Ad-hTK1-hTK1transfected in150MOI,the peak inhibitionrate reached to50.97%.Conclusion1We successfully constructed the recombinant adenovirus vector of Ad5-hTIMP1-EGFP andAd5-hTK1-hTIMP1by double promoter stragegy.2The infection rate of Ad5-hTIMP1-EGFP is in MOI-dependent (20-150MOI) andTime-dependent (1-5d) manner.3The double gene expressed recombinant adenovirus has greater inhibition effect on VSMCscompared with the single gene (hTK1and hTIMP1) adenovirus.which provides a novelstrategy for gene therapy in the prevention and treatment of vascular restenosis.