Empirical Study Of Early Growth Response Gene-1 DNA Enzyme Inhibits Intimal Hyperplasia In Autogenous Vein Graft

IntroductionBy - pass with autogenous vein graft that is generally applied modus ope-randi in Vascular Surgery, it provid a available therapeutic tool for patients of vascular obliteration. Its effect is better in the near future after operation, but in-timal hyperplasia(IH) and atherosclerosis that lead about 40% stenosis, and occlusion of vein graft 10 years after operation. Study have indicated that the functions of early growth response gene - 1 ( Egr - 1) were signal transmission in cell and participated many genie regulation, contain growth factor gene, signal conve-ry gene, transcription factor gene, oncogene and so on. It play important role to cell growth, development,differentiation and recovery. Article one illuminated that expression of Egr - 1 in vein graft, relationship of Egr - 1 and vascular smooth muscle cell(VSMC) proliferation and its effect in IH. Platelet derived growth factor - B(PDGF - B) is a growth factor intimate with VSMC proliferation and migration. Transforming growth factor - β₁, (TGF - β₁ ) is a growth factor intimate with extracellular matrix ( ECM ). Because Egr - 1 could activate transcription of many genes, so article two discussed the role of PDGF - B and TGF - β₁ in IH and relationship of they and Egr - 1 in autogenous vein graft. DNA enzyme ( DRz) is another significance discovery in life science after possess biocatalysis functional RNA that is ribozyme( Rz). The major catalytic functions of DRz include: 1) fixed - point shear RNA molecule;2) shear DNA molecule;3 ) nucleoside polyphosphate kinase activity;4 ) DNA joining enzyme activity;5 ) catalysis porphyrin genesis metal ionization. Discovery and application in gene therapy of DRz were impressed. To aim directly at Egr - 1 mRNA wedesigned Egr - 1 DNA enzyme (EDRz) in article three, as carrying agent by li-posome and discussed its inhibitory action to VSMC proliferation and intimal hy-perplasia,and confirmed the efifect of gene therapy stenosis and occlusion after vein transplantation.Materials1. Main reagents: monoclonal antibodies of Egr - 1, PDGF - B, TGF - |3, and PCNA, SABC immunohistochemical kits, in situ hybridization detection kits, RT - PCR primers of Egr - 1,PDGF - B,TGF - jj,and ï¿¡ - actin,TaKaRa kit, Egr-1 DNA enzyme (EDRz): sequence;5' - CC GCT GCC AGG CTA GCT ACA ACG ACC CGG ACG T - 3', liposome Iipofectamine? 2000.2. Main instruments:SXP - IB operating microscope,microsurgical instruments, computer image analysis system, PTC - 100TM type DNA expander, fluorescent microscopeBX60, fluorescent image analysis systemDPIO, JUNG RM2025 skiving machine,freezing skiving machine,DYY -6B and DYY - HI5 electrophoresis apparatus.Methods1. Construction of EDRz: sequence as follows: 5' - CC GCT GCC AGG CTA GCT ACA ACG ACC CGG ACG T - 3', the 3' terminus of the molecule was phosphorothioate - modificated, the 5' terminus of the molecule was marked by carboxyfluorescein(FAM), cosynthesis, 15OD260(495|xg). Added 1;1000 dieth-yl pyrocarbonate (DEPC) 80jxl, mixed centrifuge, added liposome Iipofectamine? 2000(Invitrogen) 120|xl,after lOmin added lmmol/L Mgcl232jjJ,30% F -127 Pluronic gel (Sigma) 568jxl,total 800jxl, mixed by oscillation at4tl,conserved pre - emergency in 4t refrigerator.2. Animal model;Female or male Wistar rats 90 weighing 200 ²50g offered by laboratory animal center of China Medical University, and intraperitone-al injection anaesthesia with 10% chloral hydrate solution 300mg/kg, undered the SXP - IB operating microscope ( 10 - fold) , took the right jugular vein(length 5mm) and rinsed with solution of heparin sodium, transplanted it to 3mm defect infra renal abdominal aorta with 11-0 nylon by end - end, besmeared Egr -1 DRz 8 |xl to vein graft{ including stoma) and sutured posterior peritoneum, nonuse decoagulant before or after operation.3. Indexes examined;( 1) Measured the thickness of hyperplastic intima through HE and Masson staining and computer image analysis system to evaluate the degree of vein graft intimal hyperplasia. (2)Detected the expression of Egr -1 ,PDGF - B,TGF - (5,mRNA with in situ hydridization(ISH) and reverse transcription - polymerase chain reaction ( RT - PCR ). ( 3 ) Detected the expression of Egr -1 ,PDGF - B,TGF - |$, protein with immunohistochemistry and Western blot. Detected the expression of PCNA to evaluate the degree of proliferation of VSMC in vein graft. (4) Detected the expression of EDRz mediated by liposome transfected vein graft with fluorescent microscope.Rsults1. The result of transfection of EDRz: At 1 h after graft, EDRz was mainly located media, adventitia and partial endothelial cells of vein graft. From 2h to 24h EDRz was located media of vein graft. Slight EDRz in media at 3d. EDRz was mainly located media of vein graft and found in some nascent small vessels at 7h after graft. There werent EDRz in vein grafts at 14d,28d and 42d.2. The results of histomorphology and immunohistochemistry of PCNA: Transfected EDRz, there was no obvious change of histomorphology and found minority PCNA positive cells at 2 ⁶h after graft. From 24h to 3d,we seen partial intima were damaged and amotic of endothelial cells. At 7d after graft, intima thickening and had proliferative VSMCs in it and found the expression of PCNA protein reached peak. Vein graft fundamental completed endothelialization and intimal hyperplasia reached peak at 14d, compared with no gene therapy the degree of proliferation of VSMC and thickness of intima were obviously relieved at contemporaneity. At 28d and 42d,the thickness of hyperplastic intima were decreased and the experssion of PCNA was degraded compared with no gene therapy.3. The results of RT-PCR and ISH: Egr-1 mRNA,PDGF-B mRNA and TGF - PjmRNA hadn' t been found in the normal vein,Egr - lmRNA expression had biphasic changes. At lh after graft, we found that the positive cells were located in partly VSMCs of the media, a spontaneous decline from 2h to 24h, re-induction at 3d after graft operation,a peak on day 28. The positive cells were mainly located in VSMCs of neointima. Egr - lmRNA expression hadnt biphasic changes after transfected EDRz but showed one peak, the expression of Egr - 1 mRNA reached peak at lh after operation,decreased from 2h. There werent expression of Egr -1 mRNA at 7d,14d,28d and 42d;At 6h after graft,there was the expression of PDGF - B mRNA, increased gradually from 24h to 7d,reached a peak at 14d. There was the expression of PDGF - B mRNA after transfected EDRz at 6h, reached a peak at 3d. There weren' t expression on day 28 and 42d;At early 6h after graft,there was the expression of TGF - (^mRNA, increased gradually from 24h to 3d,reached a peak at 7d. There was the expression of TGF - (^mRNA after transfected EDRz at 6h,reached a peak at 3d. There weren' t expression from 14d to 42d.4. The results of immunohistochemistry and Western blot;Egr - 1 ,PDGF - B and TGF - (3]protein hadn't been found in the normal vein. We found that Egr - 1 protein was expressed in VSMCs of media at the early phase of 2h, the expression of it was declined from 24h to 3d, reincreased at 7d, positive cells reached peak and located in VSMC of neointima at day 28. There was the expression of Egr - 1 protein after transfected EDRz at 2h, decline of its expression form 6h to 3d,no positive cells from 7d to 42d;At 6h after graft,there was the expression of PDGF - B protein and persistent increased to peak at 28 d afte graft. PDGF - B protein was expressed at 24h after transfected EDRz and persistent increased to peak at 7d. There wasnt expression of it on day 42;We detected the expression of TGF - ^ protein at 24h after graft operation and increased gradually to peak at 14d. TGF - Pj protein was expressed at 24h after transfected EDRz and persistent increased to peak at 7d. There weren' t expression of it on day 28 and 42.Conclusions1. The activition and expression of Egr - 1, PDGF - B and TGF - (â– $, are close relations with intimal hyperplasia and vascular smooth muscle cell proliferation in autogenous vein graft.2. The activition and expression of PDGF - B and TGF - (3] may depend on Egr -1 and they may contribute to the high expression of Egr -1 via a feedback mechanism in autogenous vein graft.3. Egr - 1 DNA enzyme can reduce the expression of Egr - 1, PDGF - B and TGF - (3, in autogenous vein graft.4. Egr - 1 DNA enzyme can available inhibits vascular smooth muscle cell proliferation and intimal hyperplasia in autogenous vein graft.5. Egr -1 DNA enzyme can used in prevention and therapy of angiosteno-sis and occlusion caused by autogenous vein graft.