Effect Of RNA Interference Targetting MT1-MMP On The Migration Capability Of Vascular Smooth Muscle Cell In Vitro

Partâ… The construction and identification of short hairpin RNA expression vectorof targetting rat MT1-MMP[Abstract] Objective:To construct short-hairpin RNA(shRNA)-expressingplasmid targeted to rat membrane-type 1 matrixmetalloproteinase (MT1-MMP).Methods:One pair of sense and antisense RNA oligonucleotide strands targetedto rat MT1-MMP mRNA (Accession No:NM₀31056)was designed and synthesized basedon the sequence of the rat MT1-MMP mRNA.For the preparation of duplexes,sense and antisense oligonucleotide strands were mixed together in annealingbuffer to form duplexes.Then the duplexes were cloned into the BamHâ… andHindâ…¢sites of the plasmid vector pGenesil-1,constructing a new recombinant plasmid(pGenesil-1-MT1-MMP).Finally,the recombinant plasmid was identifiedby restriction enzyme and used for sequence analysis.Results:The MT1-MMPshRNA-expression duplexes were successfully inserted into the plasmid vectorpGenesil-1,and the recombinant plasmid vectors expressing MT1-MMP-targettingshRNA were identified as our aims by restriction enzyme and used for sequenceanalysis Conclusion:Rat MT1-MMP shRNA eukaryon express plasmidvector(pGenesil-1-MT1-MMP) was successfully constructed.It laid anexperimental foundation for further researth of RNA interference therapytargetting MT1-MMP of vascular smooth muscle cell.Partâ…¡The culture and identification of vascular smooth muscle cells of ratObjective:To establish a method for culture of rat aorticvascular smooth muscle cells (VSMC)in vitro for next relatedresearches.Methods:The primary and transfer culture were done bytissue-piece inoculation and trypsin digestion.The cells were purified byseveral methods,including natural passage transfer,mechanical treatment anddifferential attachment.Morphology of the cells was observed by invertedmicroscope,and identified by immunohistochemical methods employed smoothmuscle-α-actin (α-SM-actin).Results:Ninty percent inoculated tissuepieces survived.The purity of the fifth passage SMCs was over 98%.In phasecontrast microscope,the cultured cells possessed"peak and valley"growthcharacteristics for VSMC.Immunocytochemical staining with specific mAbagainst ratα-SM-actin demonstrated these cells as positive.Conclusion:Itis a simple and reliable method for obtaining highly purified and satisfactory VSMC.It provides a good in vitro model for the researth of restenosis afterbypass grafting.Partâ…¢Effect of RNA interference Targetting MT1-MMP on the MT1-MMP gene expressionof VSMCObjective:To explore whether pGenesil-1-MT1-MMP couldeffectively transfect into VSMC and inhibit the MT1-MMP gene expression inVSMC.Methods:The recombinant plasmids were transiently transfected into VSMCcell through Lipofectamine 2000.Three groups were selected for thestudy:blank control group,negative control group and RNAi group.Forty-eighthours(48 h) after transfection,the transfection effect was confirmed byfluorescence microscope.The transfection rate was checked by flowcytometry.While 72 h after transfection,the mRNA level of MT1-MMP was detectedby semi-quantitative reverse transcription PCR(RT-PCR) and the expression ofMT1-MMP protein was detected by Western blot,respectively .Results:ThepGenesil-1-MT1-MMP plasmid vector was successfully tranfected into VSMC.Thetransfection rate at 48 h of was 85.5%.72 b after transfection ofpGenesil-1-MT1-MMP,the expression of MT1-MMP mRNA and protein wasdown-regulated significantly(P＜0.05).Conclusion:The MT1-MMP specificshRNA expression plasmid constructed in vitro was successfully transfectedinto VSMC.pGenesil-1-MT1-MMP effectively down-regulates the expression ofMT1-MMP gene. Partâ…£Effect of pGenesil-1-MT1-MMP to inhibit MT1-MMP expression on migrationcapability of VSMC in vitroObjective:To study the inhibitory effect of pGenesil-1-MT1-MMPplasmid vector on migration capability of VSMC in vitro.Methods:Therecomb(?)nant plasmids were transiently transfected into VSMC throughLipofectamine 2000.Three groups were selected for the study:blank controlgroup,negative control group and RNAi group.Migration ability of VSMC wasmeasured via Transwell chamber model and cell scratch wound model in vitro.Theproliferation ofthe cell was examined by MTT method,the apoptosis of the cellwas examined by Annexin V-FITC method.Results:After transfection ofpGenesil-1-MT1-MMP,the migration capability of VSMC decreasedsubstantially(P＜0.05).But pGenesil-1-MT1-MMP transfection had no significanteffect on the distribution of the proliferation and apoptosis of VSMC.Conclusion:pGenesil-1-MT1-MMP could decreas migration capability of VSMC invitro.