Construction Of Human Tissue Kallikrein1and Inhibitor Of Metalloproteinases1Gene Mediated By Recombinant Adenovirus Vector And Expression On Rat Vascular Smooth Muscle Cells

ObjectivesThe studies shows that tissue inhibitor of matrix metalloproteinase （TIMP1）plays an important role in the extracellular matrix formation. Preliminary studies onthis subject has found that over-expression of human tissue kallikrein1（hTK1） canpartially inhibit the proliferation of vascular smooth muscle cells and improve thevascular restenosis. However，it is not clear whether hTK1has a synergistic effect toinhibit the proliferation of the vascular smooth muscle cells with TIMP1. Firstly,recombinant adenoviral vector carrying hTIMP1gene （Ad5-hTIMP1） andco-expression recombinant adenoviral vector carrying both hTK1and hTIMP1gene（Ad5-hTK1-hTIMP1）are constructed,then the expression of （Ad5-hTK1-hTIMP1）gene on vascular smooth muscle cells （VSMCs） was tested.MethodsPartI1. After original plasmid containingh hTIMP1gene was bought fromInvitrogen company, The recombinant plasmid enhanced green fluorescentprotein （EGFP） and hTIMP1was constructed by enzyme digestion，PCRamplification，product recovery，the ligase with heat shock conversion，andits correctness is also verified by original plasmid with hTIMP1gene.2. The recombinant plasmid carrying hTIMP1and pDC316-hTK1plasmid（saved in our laboratory） was digested by selecting the appropriateendonuclease sites, and amplified gene fragment. Its product after it wasrecycled and connected was transfected to the E. Coli by the method ofheat shock,then selected the right targets from them to clone and constructedrecombinant plasmid hTK1and hTIMP1double gene,which was identified by polymerase chain reaction（PCR）, enzyme digestion and sequencinganalysis.3. The linearizing recombinant shuttle plasmid together with the plasmid wasco-transfected into the293A cells to obtain recombinant adenoviral vector.Its concentration was detected by TICD50.PartII1. Primary cultured thoracic aortic VSMCs of SHR by tissue adherencemethod,3-5generation cells selected for the experiment.VSMCs weretransfectedby three recombinant adenovirus（Ad5-EGFP,Ad5-hTIMP1-EGFPand Ad5-hTK1-hTIMP1） and then to observe the expression of the targetgene with different multiplicity of infection（MOI） and different infectiontime.2. The expression at the transcriptional level of the target gene with realtime-PCR method was determined.3. The protein expression with Western Blotting method was detected.4. The expression of the target protein with Immunofluorescence method wasdetected.Results1. The shuttle plasmid pDC316-hTIMP1-EGFP and pDC316-hTK1-hTIMP1was correctly constructed by PCR, enzyming digestion and sequencinganalysis, and successfully amplified and concentrated intoAd5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1in293A cells.The titers ofAd5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1are respectively7.0×1011vp/ml and8.5×1011vp/ml.2. The mRNA levels of target gene hTIMP1and hTK1-hTIMP1detected byreal time-PCR showed high expression after the VSMCs transfecting byAd5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1. The mRNA levels presentincrease on plural infection （50-150MOI） dependence and （1-3d） timedependence.3. The protein of target gene hTIMP1and hTK1-hTIMP1detected by Wester-blot method showed high expression after the VSMCs transfected byAd5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1. The protein presentincrease on plural infection （50-100MOI） dependence.4. Double fluoresce detect purpose gene high expression after the VSMCstransfected by Ad5-hTIMP1-EGFP and Ad5-hTK1-hTIMP1, Red express thepurpose gene protein in cells and blue marks the cell’s nucleus.Conclusion1. Use double promoter mCMV strategy, we first successfuly build andpackaging containing hTK1and hTIMP1double gene expression ofrecombinant adenovirus vector Ad5-hTK1-hTIMP1, virus drops to7.0x10¹¹vp/ml.2. After double gene recombinant adenovirus vectors infected rat VSMCs, itwas detected that the purpose genes were high expressed in transcriptionlevel and protein level.