【作者】 王欣；

【导师】 张强；

【作者基本信息】 中国医科大学 ， 外科学， 2008， 博士

【摘要】 前言以动脉粥样硬化为基础的冠心病及下肢缺血性疾病发病率逐年升高,利用自体静脉行冠状动脉搭桥术及下肢动脉旁路移植术是目前常用的治疗手段,近期疗效尚好,但由于移植静脉常常发生狭窄甚至闭塞,导致手术远期疗效较差。人们早已发现糖尿病与动脉粥样硬化有着密切的关系,近年来随着糖尿病发病率的升高,人们认识到糖尿病不但增加了患动脉粥样硬化的几率,而且使动脉粥样硬化的重要治疗手段——静脉移植术的疗效更差,其机制可能与糖尿病引起的代谢变化有关。糖尿病所带来的一个重要的代谢改变是晚期糖基化终产物(AGE)的形成,AGE一旦形成即不可逆在体内蓄积,单核-巨噬细胞的清道夫受体对其无法识别因而无法清除。近年来发现一种特殊的受体——晚期糖基化终产物受体(RAGE),它是一种完整的膜蛋白,与AGE具有高度亲和力,主要分布于血管内皮细胞、血管平滑肌细胞、肾系膜细胞及巨噬细胞等细胞表面,研究发现AGE与RAGE结合可引起多种细胞因子产生,从而引起血管内皮损伤、血管平滑肌细胞增殖、血流动力学异常以及细胞基质异常增生等一系列病理改变。核因子-KappaB(NF-κB)的激活是最重要的信号传导途径。氨基胍是一种具有亲核作用的肼化合物,能够减少AGE的产生并抑制AGE与胶原蛋白及白蛋白等蛋白糖化后的交联,从而阻断AGE与RAGE的结合。本实验论文一通过检测RAGE在糖尿病大鼠自体移植静脉中的表达及移植静脉内膜增生情况和平滑肌细胞增殖情况,探讨RAGE与糖尿病大鼠移植静脉内膜增生的关系;论文二通过检测RAGE、NF-κBp65和血管细胞黏附分子-1(VCAM-1)在自体移植静脉中的表达、血清AGE含量及移植静脉内膜增生和平滑肌细胞增殖情况,探讨糖尿病大鼠移植静脉内膜增生加重的机制;论文三通过氨基胍干预并检测RAGE、NF-κB p65在自体移植静脉中的表达、血清AGE含量及移植静脉内膜增生和平滑肌细胞增殖情况,探讨阻断AGE-RAGE结合对糖尿病大鼠移植静脉内膜增生的影响,从而为临床提供一个可能的治疗靶点。实验材料与方法一、实验材料1、实验动物雄性Sprague-Dawley(SD)大鼠,体重250—280克,中国医科大学实验动物中心提供。2、主要试剂链脲佐菌素(streptozotocin,STZ)、盐酸氨基胍(aminoguanidine,AG)、抗RAGE多克隆抗体、抗NF-κB p65多克隆抗体、抗VCAM-1单克隆抗体、抗PCNA单克隆抗体、SP试剂盒、DAB显色试剂盒,Trizol试剂、RT-PCR试剂盒、RAGE、NF-κB p65、VCAM-1及内对照GAPDH的PCR引物等。3、主要仪器Accu-Chek血糖仪、SXP-1B型双人双目手术显微镜、显微外科手术器械、-70℃深低温冰箱、JUNG RM2025切片机、显微镜(带相机)、TGL-16B台式高速离心机、722分光光度仪、紫外分光光度仪、850荧光分光光度仪、DYY-6B和DYY-Ⅲ5型电泳仪、Rotorgene2000基因扩增仪,计算机图像分析系统等。二、实验方法1、建立糖尿病大鼠模型配制STZ枸橼酸缓冲液溶液,浓度1%,pH4.0-4.5。大鼠禁食24h后,行单次STZ腹腔注射(60mg/kg),48h后大鼠由尾静脉取血测定非空腹血糖,超过16.7mmol/L为模型建立成功标准。2、建立大鼠静脉移植模型大鼠腹腔注射10%水合氯醛水溶液(300mg/kg)麻醉,固定后在SXP-1B型显微镜(10倍)下行无菌显微外科手术:取大鼠右颈总静脉长约0.8cm,肝素盐水冲洗后,用11-0血管缝合线端-端吻合移植于肾下腹主动脉,每个吻合口缝合12—16针,确定血管通畅后关闭腹腔,术中及术后均不使用抗凝剂,术后常规肌肉注射抗生素三天。3、标本的获取大鼠腹腔注射10%水合氯醛水溶液(300mg/kg)麻醉,原切口进入,结扎后取下移植静脉段,肝素盐水冲洗,另取大鼠左侧颈总静脉作为对照,另经腹主动脉取血0.5ml后大鼠放血处死。每个移植静脉标本分为两段,一半液氮冻存,另一半置于4%中性甲醛溶液固定24h后常规脱水、透明、浸蜡、包埋制成蜡块。4、指标检测(1)石蜡切片行HE染色和Verhoeff弹力纤维染色,辅以计算机图像分析系统,测量增生内膜厚度及内膜/中膜厚度比,评估移植静脉内膜增生程度。(2)石蜡切片行免疫组织化学方法检测PCNA表达,评估移植静脉平滑肌细胞增殖程度。(3)免疫组织化学方法和Western蛋白印迹方法检测移植静脉RAGE、NF-κBp65及VCAM-1的蛋白表达情况。(4)逆转录聚合酶链式反应(RT-PCR)方法检测移植静脉RAGEmRNA、NF-κB p65mRNA及VCAM-1mRNA的表达情况。(5)荧光分光光度法测定大鼠血清AGE含量。5、统计学分析所有数据以(?)±s表示,组间比较采用t检验或单因素方差分析,所有统计计算由软件SPSS 13完成,P＜0.05为差异有统计学意义。实验结果1、组织形态学及PCNA免疫组化结果糖尿病大鼠正常静脉内膜为单层内皮细胞,中膜由2～3层平滑肌细胞及胶原组成。静脉移植术后随着时间延长,移植静脉管腔新生内膜出现并逐渐增厚,内膜/中膜厚度比值显著增加;PCNA阳性细胞百分率术后24h开始逐渐增加,但术后28d后开始减少。术后7d及14d,与非糖尿病大鼠比较,糖尿病大鼠移植静脉内膜厚度、内膜/中膜厚度比及PCNA阳性细胞百分率均增加,差异有统计学意义(P＜0.05);给予氨基胍干预后,糖尿病大鼠移植静脉内膜厚度、内膜/中膜厚度比及PCNA阳性细胞百分率均较无氨基胍干预的糖尿病大鼠减少,差异有统计学意义(P＜0.05),而与非糖尿病大鼠相比,差异无统计学意义(P＞0.05)。2、免疫组织化学方法和Western蛋白印迹方法检测结果糖尿病大鼠正常静脉未见RAGE蛋白表达,静脉移植术后24h开始出现移植静脉的RAGE蛋白表达,术后3d开始增加明显,术后28d后增加速度减缓。与非糖尿病大鼠相比较,糖尿病大鼠移植静脉RAGE、NF-κB p65及VCAM-1的蛋白表达均明显增加,差异有统计学意义(P＜0.05)。给予氨基胍干预后,糖尿病大鼠移植静脉NF-κB p65蛋白表达较无氨基胍干预的糖尿病大鼠减少,差异有统计学意义(P＜0.05),而与非糖尿病大鼠相比,差异无统计学意义(P＞0.05)。给予氨基胍干预后,糖尿病大鼠移植静脉RAGE蛋白表达与无氨基胍干预的糖尿病大鼠比较,差异无统计学意义(P＞0.05)。3、RT-PCR结果糖尿病大鼠正常静脉未见RAGEmRNA表达,静脉移植术后6h开始出现移植静脉的RAGEmRNA表达,术后24h开始逐渐增加,术后28d后增加速度减缓。与非糖尿病大鼠相比较,糖尿病大鼠移植静脉的RAGEmRNA、NF-κB p65mRNA及VCAM-1mRNA表达均明显增加,差异有统计学意义(P＜0.05)。给予氨基胍干预后,糖尿病大鼠移植静脉NF-κB p65mRNA表达较无氨基胍干预的糖尿病大鼠减少,差异有统计学意义(P＜0.05),而与非糖尿病大鼠相比,差异无统计学意义(P＞0.05)。给予氨基胍干预后,糖尿病大鼠移植静脉RAGEmRNA表达与无氨基胍干预的糖尿病大鼠比较,差异无统计学意义(P＞0.05)。4、血清AGE含量测定结果糖尿病大鼠血清AGE含量显著高于非糖尿病大鼠(P＜0.05)。给予氨基胍干预后,糖尿病大鼠血清AGE含量明显减少,与无氨基胍干预的糖尿病大鼠比较,差异有统计学意义(P＜0.05),与非糖尿病大鼠相比,差异无统计学意义(P＞0.05)。结论1、RAGE的表达增加与糖尿病大鼠自体移植静脉内膜增生关系密切。2、AGE与RAGE结合后激活NF-κB,接下来使黏附分子释放,最终导致血管平滑肌细胞增殖与迁移,这可能是糖尿病加重自体移植静脉内膜增生的机制。3、氨基胍可通过抑制AGE的产生来阻断AGE-RAGE结合从而有效减轻糖尿病自体移植静脉的内膜增生。4、RAGR可能成为糖尿病自体移植静脉内膜增生的治疗靶点。更多还原

【Abstract】 IntroductionCoronary atherosclerotic heart disease and lower limbs arteriosclerosis are critical diseases in the senior population and the use of vein grafts as bypass conduits is one of most effective treatments.Although vein grafts are successful in relieving symptoms in patients who suffer from severe ischemic arterial disease,long-term patency is still a critical problem hampered by intimal hyperplasia,a common response of a vein after grafted into the arterial circulation.It is well known that diabetes mellitus is associated with atherosclerosis.Diabetes mellitus is not only a risk factor for atherosclerosis,but is also associated with worse outcomes when the saphenous vein graft is used in coronary and infrageniculate reconstruction.Under diabetic conditions,advanced glycation end products（AGE） is known to accumulate at an extremely accelerated rate both in various tissues and in the circulation and has been implicated in the development of diabetic vascular complications.Receptor-dependent mechanisms are likely to be involved in the AGE-induced tissue dysfunction.The receptor for advanced glycation end products（RAGE） is a multi-ligand cell surface receptor belonging to the immunoglobulin super-family of cell surface molecules.RAGE consists in vascular endotheliocyte,vascular smooth muscle cell,and monocyte.The engagement of RAGE by AGE can induce cellular oxidant stress,attract inflammatory cells such as polymorphonuclear leukocytes,mononuclear phagocytes,and T-lymphocytes into the tissue provides a mechanism to sustain the inflammatory response and result in the injury of vessel.In this process,the activation of nuclear factor-kappa B（NF-κB） is the most important signal transmission pathway. Aminoguanidine is a kind of hydrazine compound which has nucleophilic function and can block the early glycation production from rearranging and dehydrating to form AGE,thus reducing the production of AGE.Article one illuminated the expression of RAGE in vein graft of diabetic rats and the relationship of RAGE and intimal hyperplasia.By examining the expression of RAGE,NF-κB p65 and vascular cell adhesion molecule-1（VCAM-1） in vein graft of diabetic rats,article two aimed at exploring the mechanism of vein graft intimal hyperplasia increasing of diabetic rats. Article three is to explore the modificatory effect to vein graft intimal hyperplasia of diabetic rats by blocking the engagement of AGE-RAGE.Materials and MethodsMaterials1.Animals:Sprague Dawley rats（body weight ranged from 250g to 280g） were obtained from laboratory animal centre of China Medical University.2.Main reagents:streptozotocin（STZ）,aminoguanidine（AG）,polyclonal antibody against rat RAGE and NK-κB p65,monoclonal antibody against rat VCAM-1 and PCNA,SP immunohistochemical kits,Trizol reagent,RT-PCR kit,RT-PCR primers of RAGE,NK-κB p65,VCAM-1 and GAPDH（reference）.3.Main instruments:Accu-Chek blood glucose monitoring system,SXP-1B operating microscope,microsurgical instruments,-70℃deep low temperature refrigerator,JUNG RM2025 skiving machine,microscope with camera,TGL-16B high speed centrifugal,722 spectrophotometer,ultraviolet spectrophotometer,850 fluorospectrophotometer,DYY-6B and DYY-Ⅲ5 electrophoresis apparatus, Rotorgene2000 gene amplifier,computer image analysis system.Methods1.Diabetic animal:Fasting 24h,the rats were given a single intraperitoneal injection of 60 mg/kg of STZ dissolved in freshly prepared 0.1mol/L citrate buffer （pH4.0-4.5） immediately before administration.Forty-eight hours later,the blood glucose levels were sampled from tail veins and measured with Accu-Chek blood glucose monitoring system to ensure induction of diabetes（blood glucose≥16.7mmol/L）.2.Rat venous intimal hyperplasia model:Rats were anesthetized by 10%chloral hydrate solution,i,p.injection,300 mg/kg.The right external jugular vein（≈0.8 cm） was grafted into the infrarenal abdominal aorta with 11/0 suture under SXP-1B operating microscope（10-fold）.Twelve to sixteen interrupted sutures were required per end.The aorta was clamped for an average of 60 min.Ensuring the vein graft’s patency before closing the muscle and skin.The rats were given intramuscular injection next three days.3.Tissue harvest:Vein grafts were harvested after the rats were anesthetized,the left external jugular veins were harvested as a control at the same time.Before rats were sacrificed,0.5 ml blood was collected from the abdominal aorta.Each sample was divided into two parts from center,proximal half was embedded in paraffin for morphological measure and distal half was freezed by liquid nitrogen for Western blot and reverse transcription-polymerase chain reaction（RT-PCR） analysis.4.Indexes examined:（1） Measured the thickness of hyperplastic intima though HE and Verhoeff staining and computer image analysis system to evaluate the degree of vein graft intimal hyperplasia.（2） Detected the expression of PCNA with immunohistochemistry to evaluate the degree of proliferation of VASC in vein graft.（3） Detected the expression of RAGE,NK-κB p65 and VCAM-1 protein with immunohistochemistry and Western blot.（4） Detected the expression of RAGE,NK-κB p65 and VCAM-1 mRNA with RT-PCR.（5） The serum AGE level was determined by fluorospectrophotometry.5.Data were expressed as mean±S.D.Means of different groups were compared with analysis of variance（ANOVA） and t-test.P＜0.05 was considered to indicate statistical significance.Results1.The results of histomorphology and immunohistochemistry of PCNA:The thickness of hyperplastic intima in vein graft of diabetic rats was increased with time after operation.The PCNA positive cells appeared at 24h after operation,rised to peak at 28d after operation and falled later.After 7 or 14 days surgery,the thickness of hyperplastic intima and the ratio of intima to media thickness in diabetic rats were significantly greater than non-diabetic rats（P＜0.05）.Interesting,the thickness of hyperplastic intima and the ratio of intima to media thickness in diabetic rats drunk AG were significantly less than diabetic rats drunk distilled water（P＜0.05） and had no difference compared with non-diabetic rats（P＞0.05）.Similar results were obtained for PCNA positive cell percentage.2.The results of immunohistochemistry and Western blot:The expression of RAGE was not found in normal vein of diabetic rats and appeared at 24h after operation in vein graft of diabetic rats.The expression of RAGE in vein graft of diabetic rats was increased with time,which increased slowly after the 28^th day.After 7 or 14 days surgery,the percentage of RAGE,NK-κB p65 and VCAM-1 positive cells in diabetic rats was significantly increased compare to non-diabetic rats（P＜0.05）.There is no significant difference between diabetic rats drunk AG or distilled water in the percentage of RAGE positive cells（P＞0.05）.The percentage of NF-κB p65-positive cells in diabetic rats drunk AG was significantly decreased compared to diabetic rats drunk distilled water after 7 and 14 days surgery（P＜0.05）,and no difference compare to non-diabetic rats（P＞0.05）.3.The results of RT-PCR:There was not the expression of RAGE mRNA in normal vein of diabetic rats.The expression of RAGE mRNA appeared at 24h after operation in vein graft of diabetic rats and till 28d.Later the rise was slow.After 7 or 14 days surgery,the expression of RAGE,NK-κB p65 and VCAM-1 mRNA in diabetic rats was significantly increased compare to non-diabetic rats（P＜0.05）.There is no significant difference between diabetic rats drunk AG or distilled water in the expression of RAGE mRNA（P＞0.05）.The expression of NF-κB p65 mRNA in diabetic rats drunk AG was significantly decreased compared to diabetic rats drunk distilled water after 7 and 14 days surgery（P＜0.05）,and no difference compare to non-diabetic rats（P＞0.05）.4.The results of serum AGE:The serum AGE level in diabetic rats was significantly more than which in non-diabetic rats（P＜0.05） 7 or 14 days after operation.After 7 and 14 days grafting,the serum AGE level in diabetic rats that dnmk distilled water significantly increased compared to drunk AG（P＜0.05）.However, there is no significant difference between diabetic rats that drunk AG and non-diabetic rats（P＞0.05）.Conclusion1.Upregulation of RAGE expression has a close relationship with intimal hyperplasia and vascular smooth muscle cell proliferation in autogenous vein graft of diabetic rats.2.The engagement of RAGE by AGE activated the NF-κB,which increased the release of adhesion molecule;the next consequence is the proliferation of vascular smooth muscle cell.It maybe the mechanism of vein graft intimal hyperplasia increasing of diabetic rats.3.Aminoguanidine can inhibit the intimal hyperplasia of diabetic rats by blocking the engagement of RAGE by AGE.4.RAGE can become an effective target for preventing the development of intimal hyperplasia.更多还原