Effect Of MMP-3 5A/6A Polymorphism And It Inhibitor TIMP-1 In Restenosis After PCI

Objective:In order to find the relationship of MMP-3 polymorphism and restenosis after PCI,than try to attempt a way to decrease restenosis rate by gene therapy.The affections of MMP-3 promoter 5A/6A polymorphism on gene expression and regulation and TIMP-1 function on restenosis after rat carotid artery injury were observed in out study.Methods:261 patients were selected in our study who repeats angiography in our hospital during the period of 2005 and 2007.Time of first to second angiography interval was 24.83±4.11 months.Patients were divided into control group(n=105)and restenosis group(n=156)according to the angiography result.And we also collected the basic data to found if it do some affection on restenosis.Each patient DNA was extraction from blood.The common polymorphism in the MMP-3 gene promoter (5A/6A)was studied through polymerase chain reaction(PCR)amplification with the use of mutagenic primers.A 130-bp fragment was amplified from genomic DNA with use of the special primers.Thus,a recognition site for the enzyme Tth111I (GACN/NNGTC)is created in the case of polymorphism.According the result 261 patients were divided into wild type(6A/6A)(n=180)and mutant type (5A/5A+5A/6A)(n=81)to investigated the relationship of MMP-3 polymorphism and restenosis.Use molecular cloning techniques,MMP-3 promoter fragment 5A/5A or 6A/6A allele were cloned into plasmid of pUCm-T vector to construct pUCm-T/5A and pUCm-T/6A recombinant plasmid.After use restriction enzyme to confirm the sequence is correct than recombinant plasmid of pGL2-Basic/5A and pGL2-Basic/6A. Use the technique of cation lipidosome transfection,recombinant plasmid was transfercted into HepG2 cell.After cell were cultured in vitro,collected the cells to detect the intensity of luciferase.In animal test 36 female SD rat were used in our study.Each rat was injured left common carotid artery by sofe needle from left external carotid artery.And than random decided into three groups,blank control group(do not give any intervention factors),AdCMV group(control group,only give adenovirus transfection)and AdhTIMP-1 group(experimental group,just give human TIMP-1 gene delivery by adenovirus).After operation,three group were given high fat diet.2 weeks later,every rat left carotid artery was obtained to HE dying and immunohistochemistry analysis.Results:Use Logistic analysis we found target vascular type(ACC/AHA), reference diameter and stent type(bare metal stent or drug-eluting stent)were significant difference in control and restenosis group(p=0.040,0.006,0.007). Incidence of restenosis difference between the group of wild type and mutant type was failure to find.But accuracy in wild type group the grade of remodeling was high than mutant type.And from another point the patient who carried 6A allele had more incidence happened restenosis than 5A allele.In vitro PCR and double restriction enzyme digestion showed that the recombinant vectors were constructed correctly. These vectors were highly expressed in the HepG2 cell.The 6A/6A genotype showed lower expression of luciferase reporter compared to the 5A/5A genotype.Animal test showed 2 weeks after gave high fat diet,CHO,TG,LDL-C and HDL-C were significant difference than normal.HE dyeing only seen tunica media a bit of thicker in blank control and control group.But immuneohistochemistry analysis seen MMP-3 content in the area of injury significant higher in blank and control group than in experimental group.And TIMP-1 content significant higher in experimental group than other groups.Conclusion:The patient who placed the bare-metal stent in small vascular (＜3mm)with C type of vascular lesion(ACC/AHA classification)more easily occur restenosis and MMP-3 gene 5A/6A polymorphism maybe some useful on predictive of vascular remodeling.The fragment of MMP-3 promoter which contain -1612 point polymorphism 6A allele gene expression is lower than 5A allele.So we suppose this is maybe one of the reasons for MMP-3 polymorphism participated a role in restenosis after PCI.Human TIMP-1 delivered by adenovirus can successfully transfected into rat carotid artery vessel smooth muscle cell and interfered in restenosis after vessel injury by soft needle.So we can suppose that gene therapy by adenovirus delivery hTIMP-1 maybe a new way to restenosis rate.