【作者】 周敬；

【导师】 刘闺男；

【作者基本信息】 中国医科大学 ， 内科学， 2005， 硕士

【摘要】 前言 经皮冠状动脉介入治疗（percutaneous coronary intervention,PCI）是公认的治疗冠心病的有效方法之一,但术后6个月内再狭窄发生率仍可达到20%-30%,严重地影响了该项治疗技术的远期疗效。目前认为,动脉平滑肌细胞的过度增生是PCI术后再狭窄的重要病理特征。Egr-1是立早基因（IEGs）家族的一员,是一种重要的核转录因子,调控着多种与细胞增殖相关基因的表达,因此与细胞增殖关系密切。 脱氧核酶（deoxyribozyme,DRz）是具有酶活性的DNA分子,DRz10-23是其中的一种,它由15个脱氧核苷酸构成催化特异性结构域,两边分别连有7-8个脱氧核苷酸构成底物识别结构域,RNA底物通过Waston Crick碱基配对形式与酶结合,未配对的嘌呤和配对的嘧啶之间构成特异性磷酸二酯键切割位点,从而将底物RNA切断。本研究设计了针对大鼠Egr-1的特异性脱氧核酶DRz10-23（ED5）,作用于损伤后的动脉内膜,观察其对大鼠颈总动脉损伤后内膜增生的影响,探讨其作用机制。 实验方法 2F Fogarty导管对雄性Wistar大鼠行颈总动脉损伤术后,在转染试剂Fu gene 6介导下经血管腔内转染Egr-1的特异性脱氧核酶（ED5）,以假手术组、单纯损伤组及空白转染试剂组（Fu gene 6组）作为对照,于术后3、7、14和21天4个时间点取材,检测内膜增生程度及增殖细胞核抗原（PCNA）、转化生长因子-β₁（TGF-β₁）和Egr-1的表达变化。结果采用SPSS10.0统计软件处理,多组之间的两两比较用q检验,P<0.05则认为统计学上差异有显著性。更多还原

【Abstract】 IntroductionPercutaneous coronary intervention is regarded as one of useful treatments for coronary atherosclerosis heart disease, however , it is hampered by local arterial restenosis, which occurs in 20 - 30% of cases within 6 months. Vascular smooth muscle cells hyperplasia is important pathological characters of post percutaneous coronary intervention restenosis, Early growth response factor - 1 , a transcription factor, is a member of immediate - early genes family that regulates a number of pathophysiologically relevant genes, so it is associated with cell proliferation.Deoxyribozymes are catalytic antisense DNA molecules. DRz10 - 23, one of them, comprises a catalytic domain composed of 15 deoxynucleotides ,flanked by two substrate recognition arms each composed of 7 - 8 deoxynucleotides that bind to target RNA through Watson - Crick base - pairing . The DNAzyme cleave a specific phosphodiester linkage between an unpaired purine （ A, G） and a paired pyrimidine （ C, U）. Here we designed DNA enzyme targeting Egr - 1 mRNA of rats and then transfected to the rats common carotid arteries subjected to balloon injury to study the effects on neointimal formation of rat common carotid arteries.MethodsThe male Wistar rats common carotid arteries subjected to 2 F Fogarty bal-loon injury were immediately transfected with ED5 by Fu gene 6 from lumen, sham group 、 balloon injury alone group and Fu gene 6 group served as control groups. Arteries were harvested at day 3、7、14 and 21 after surgery. The extent of intimal hyperplasia and change of expression of proliferating cell nuclear antigen （ PCNA）、 transforming growth factor - β₁ （TGF - β₁） and Egr - 1 were detected . The results are dealed with SPSS 10. 0. Significant assay uses q - test, and the value of P <0.05 is considered significant.ResultsThe intimal denudeation was seen after rats common carotid arteries were subjected to balloon injury , it proved that the animal models of artery injury had been established successfully. Vascular intima was completely covered with cells and intimal hyperplasia was not seen in sham group. The intimal thickness existed at day 7 in arteries of Fu gene 6 group 、injury alone group and it was more significant at day 14、21, there was a number of proliferation cells in neointimal and medial layers, internal elastic layers were uncleared. There was no significant difference between them. Compared with balloon injury alone group at the same time , intimal hyperplasia of ED5 group was inhibited, there were significant differences between ED5 group and injury alone group （ P <0. 01）.The expression of PCNA was not detected and TGF - β₁ and Egr - 1 were expressed at low levels in the normal artery. The positive expression of PCNA and TGF - β₁ reached their peaks in neointimal and medial layers at day 7 and began decreasing at day 14 after injury , but the expression of Egr - 1 was persistent. In arteries treated with ED5 , the expression of PCNA、TGF -β₁ and Egr -1 reduced at the same time point. Compared with balloon injury alone and Fu gene 6 group,there were significant differences（ P <0.001）.DiscussionPercutaneous coronary interventions one main method of resolving ischemia - reperfusion hearts, however, arterial walls are injuried unavoidly during mul-tipulation of using by cather, once it occurs, various transcription control genes, such as Egr -1, will be activated that might contribute to the smooth cells hyperplasia and crine extracellular matrix , thick neointimal will be become fanial-ly. We hypothesizes that smooth cells hyperplasia might be attenuated and thus intimal thickness be decreased by inhibiting the expression of these relevant genes.Deoxyribozymes’finding is a magnificient development after ribozymes. Man realizes that DNA molecular can not only store heredity information but also has biological catalytic activities. Compared with ribozymes, Deoxyribozymes have many advantages : stable property 、 simple instruction、 good accessibility to sub-strate、more cleaving targets to be choosen、higer sequent - specificity. At present, the studies at home are limited to virology, especially in hepatitis virus treatment, but fewer in vascular proliferation disease. Here we designed rats’ DNA enzyme targeting Egr -1 mRNA and then transfected it to the rats common carotid arteries subjected to balloon injury . The results showed that the expression of PCNA 、TGF -β₁ and Egr - 1 reduced and intimal hyperplasia decreased, it suggested that ED5 could attenuate intimal hyperplasia after ballon by specially suppressing the expression of PCNA、TGF -β₁ and Egr -1. It is known that there are two bind zones of Egr - 1 in TGF -β₁ promoter regions by which active Egr - 1 could control TGF - β₁ expression, so Egr - 1 plays a key role in cell proliferation.The main difficulty of gene treating restenosis is that oligodeoxynucleotide is degraded by exonuclease or dismissed with blood before entering into nuclei . To prove the effection of treatment, the Wistar rats common carotid arteries subjected to balloon injury were immediately transfected with ED5 by Fu gene 6 from lumen. We choose Egr - 1 as a ideal target for therapeutic intervention to prevent restenosis because Egr -1 is a important transcription factor which associates stimulus with signal transduction system and mediates the expression of various genes. So many relevant genes expression are controlled by regulating Egr -1. It could be more efficient than inhibiting only one gene method. Nowaday it is reported that inhibition of neointimal hyperplasia after ballon injury by cis - element decoy of early growth response gene -1 in hypercholesterolemic rabbits. It更多还原