【作者】 王瑞华；

【导师】 金星；

【作者基本信息】 山东大学 ， 普通外科学， 2014， 博士

【摘要】 背景对长段或多节段中小血管慢性阻塞而不适于血管腔内干预的患者,采用血管旁路术行冠状动脉或肢体动脉转流手术,目前仍然是挽救患者生命和肢体功能的主要手段。自体静脉是血管旁路移植物的金标准,与其他类型的移植物相比,自体静脉最大的特质就是静脉血管术后对于动脉环境的适应性改变,这种适应性改变通常被称为“静脉动脉化”。自体静脉移植物术后动脉化的能力是其他人造移植物无法替代的。从1950年开始自体静脉被广泛应用于缺血性疾病患者的血管重建手术,但是20-50%的静脉移植物由于其手术以后的重塑过程导致血栓形成、再狭窄等,最终导致血管移植手术失败。一旦血管重建术后再狭窄形成,就需要再次进行血管移植术或血管内支架置入术等血管重建手术,手术引起的血管损伤,日后仍会导致血管再狭窄形成。目前仍没有有效的治疗方法来减轻移植静脉血管壁的过度增厚,移植血管再狭窄的机制及其防治措施一直是临床研究的热点。从目前的研究结果来看,移植静脉早期再狭窄与缺氧、手术过程损伤等原因导致的血管内皮损伤部位急性血栓形成有关；移植静脉中后期再狭窄与中膜血管平滑肌细胞、外膜成纤维细胞的迁移与增殖、细胞增生与凋亡失去平衡、新生内膜过度增生密切相关。血管平滑肌细胞过度增殖并向内膜迁移是新生内膜增生的重要病理基础,是各种因素导致血管重建术后血管再狭窄的关键环节。因此,抑制血管平滑肌细胞过度增殖及迁移是防治静脉移植术后再狭窄的有效策略。移植静脉内膜增生的发生过程是多种细胞因子和血管活性物质介导的局部血管重构的过程,涉及到一系列复杂的病理过程,目前临床使用的药物都未能有效降低再狭窄的发生率。基因转移技术是防治移植静脉新生内膜过度增生的重要方法,选择适当的靶基因进行局部转染表达,针对血管再狭窄不同时期病变的进程,抑制内膜增生的病理过程。国内外学者已经作了许多这一方面的研究,但目前尚未找到一种满意的移植血管基因转移方法,实际应用时如何选择相应的血管基因转移技术(选取高效低毒的载体、提高转染率、减轻副作用等),可能是今后研究的方向之一。纳米基因载体是将DNA.RNA等治疗分子包裹在纳米颗粒之中或吸附在其表面,在细胞摄取作用下进入细胞内,实现目的基因持续稳定的表达。可生物降解的PLGA(乳酸-乙醇酸共聚物)纳米粒载药系统因其具有缓释作用而成为纳米粒子研究中的热点。但是由于治疗基因多是高度亲水的大分子物质,将其包封于疏水性的PLGA纳米粒中存在一定困难,而且DNA从疏水性的PLGA纳米粒中的释放过于缓慢,从而影响DNA的起效时间,因而有必要对PLGA进行结构修饰以提高其亲水性,以改善载基因纳米粒的包封率和释放性能。Arresten基因是2000年由Colorado等研究发现的一种基底膜来源的内源性血管生成抑制因子,由230个氨基酸残基组成,其分子量约为26kD,是Ⅳ型胶原a1链的羧基末端NCl结构域多肽片段。研究证实,arresten抑制新血管形成及肿瘤生长和转移效果明显,其活性为现今热点研究中的Endostatin的3-10倍,因此,arresten被认为是继Endostatin之后新的极具潜力的血管生长抑制因子。arresten在IV型胶原聚集、基底膜形成、调节细胞生物学行为方面发挥重要作用,能抑制内皮细胞、血管平滑肌细胞的增殖和迁移,诱导内皮细胞的凋亡。综合既往关于血管移植术后再狭窄和基因治疗及纳米载体研究的理论成果和结论,我们提出一个新的关于血管移植术后再狭窄治疗的假设：把arresten基因治疗和纳米粒载体系统两种方法联合起来,利用经过PEG修饰的PLGA纳米粒子介导arresten基因局部转染移植静脉,增强基因耐核酸酶的能力,改善载基因纳米粒的包封率和释放性能,延长纳米粒在体内血液循环的时间。课题根据这一个假设进行设计,拟明确纳米粒介导arresten基因转染对移植静脉内膜增生的影响,并研究其发挥作用的机制,从而为临床提高血管重建术的中远期疗效提供治疗思路。目的研究构建arresten基因真核表达载体；以PEG-PLGA为载体材料,制备出生物可降解型载基因纳米粒；建立兔自体静脉移植模型,局部定位转染arresten基因纳米粒,通过移植静脉病理图像分析、病理组织学观察、免疫组化分析、荧光定量PCR等手段,探讨纳米粒介导人arresten基因局部转染对兔移植静脉内膜增生的作用及其可能的作用机制。方法(1)设计arresten序列进行人工合成,根据构建需求在5’端添加ATG起始密码子,并添加增强表达的kazak序列GCCACC3’端去除终止密码子,共计708bp,并在上游引入Hind Ⅲ酶切位点,下游引入BamH Ⅰ酶切位点。序列合成后装载至pUC57中间载体,并进行酶切及测序验证。双酶切pUC57-arresten和pEGFP-N1质粒,在T4DNA连接酶的作用下进行连接反应。采用内切酶BamH Ⅰ和EcoR Ⅰ进行双酶切鉴定重组质粒pEGFP-N1-arresten。取纯化的重组质粒样品,进行核苷酸序列测定,以确认目的基因序列是否正确。(2)采用改良的纳米粒沉淀法制备包封型载arresten基因PEG-PLGA纳米粒。称取20mg PEG-PLGA,接着将其溶解在lml DMSO的溶液中(含200μg的DNA),然后把此溶液在微量注射泵的配合下滴入20m1的F127(0.5%,w/v)的乳化剂中以制备载基因的纳米粒混悬液。最后,混悬液经低温高速的离心作用后,收集其离心上清液应用PicoGreen荧光分光光度法测定载基因纳米粒的包封率及理化性质。(3)以新西兰大白兔为研究对象,动物随机分为3组：空白组、对照组、arresten组,每组10只。采用游离的颈外静脉间位移植至颈总动脉的方法建立兔自体静脉移植模型。在行血管吻合术前,将移植段血管置于纳米粒介导的重组质粒pEGFP-N1-arresten溶液中浸泡,转染移植静脉。术后1周及术后4周分别应用彩超观察静脉桥通畅情况。手术4周后取出移植静脉,常规行HE及Masson染色,运用计算机图象分析技术来检测兔移植静脉的内膜与中膜的厚度；采用免疫组化的方法来检测金属基质蛋白酶-2(MMP-2)、金属基质蛋白酶-9(MMP-9)、平滑肌细胞肌动蛋白(a-SMA)、细胞增殖核抗原(PCNA)的阳性表达情况,荧光定量PCR方法检测移植静脉中arresten基因mRNA转录水平。结果(1)重组质粒序列比对分析显示,插入到表达载体的目的基因的序列与人arresten基因片段序列完全一致,目的基因序列正确,证明arresten基因准确的克隆至表达载体中,试验成功地构建出arresten基因真核表达载体pEGFP-N1-arresten。(2)试验顺利制备了一种包封型的载arresten基因的PEG-PLGA纳米粒。这种纳米粒呈均匀的球形,纳米粒的粒径分布不宽,arresten基因能够高效的包封于PEG-PLGA纳米粒中(包封率96.64±0.202%),DNA的结构得到保持。(3)成功建立兔自体静脉移植模型30只,手术过程无死亡,1只术后20天死于消化道感染,余均健康存活。29只存活动物手术后无活动功能障碍,术后精神、饮食、活动正常,切口愈合好,无红肿、渗出或脓肿形成。取材前检查存活动物移植静脉27例通畅,静脉搏动良好,血管与周围组织有粘连,未见移植静脉破裂,无深部感染,1例移植静脉重度扩张导致血管瘤形成,2例闭塞。术后第1、4周分别观察到1只动物移植静脉闭塞。切取标本行荧光定量PCR,结果显示重组质粒pEGFP-N1-arresten转染的血管组织中有目的基因mRNA的表达；通过病理计算机图象技术分析可知,转染arresten纳米粒组的内膜厚度以及中膜厚度均分别比对照组、空白组小,转染arresten纳米粒组的内膜增生的程度分别比空白组、对照组轻(P<0.05)。血管平滑细胞是增生内膜中主要的细胞；转染arresten纳米粒组的移植静脉的PCNA阳性细胞数量与表达指数,均比空白组、对照组更小(P<0.05)；转染arresten纳米粒组的MMP-2及MMP-9表达均少于空白组及对照组(P<0.05),差异有统计学意义。结论采用改良的纳米粒沉淀法成功制备了包封型载pEGFP-N1-Arresten基因PEG-PLGA纳米粒,质粒DNA可以行之有效地包封在PEG-PLGA纳米粒里面(研究结果显示,这种包封率超过95%),也较好地保持了DNA结构的完整性。采用游离的颈外静脉间位移植至颈总动脉的方法可以成功建立兔自体静脉移植模型,具有成功率高、损伤小、围手术期死亡率低等优点。采用移植静脉浸泡于基因纳米粒中的方法可以成功将目的基因导入移植静脉细胞中,PEG-PLGA纳米粒可以介导局部定位转染人arresten基因,从而有效抑制兔移植静脉内膜的增生,人arresten基因抑制兔移植静脉内膜的增生的作用机制,可能是通过抑制MMP-2、MMP-9的表达,从而抑制血管平滑肌细胞的迁移和增殖。人arresten基因在预防静脉动脉化过程内膜过度增生方面具有很好的临床前景,其对血管壁各种细胞迁移及增殖的作用,以及其抑制移植静脉内膜增生的具体信号传导机制等有待进一步探讨。更多还原

【Abstract】 BACKGROUND For patients with coronary artery disease or limb ischemia, placement of a vein graft as a conduit for a bypass is an important and generally durable strategy among the options for arterial reconstructive surgery. Autogenous Vein is the gold standard for vascular graft conduits. Compared with other types of graft conduits, the most distinctive property of the vein graft is the adaptive response to the arterial environment during the post-surgical process; this adaptation is thought to be responsible for the superior performance of vein grafts compared with prosthetic grafts. However, it is also known that abnormal, or uncontrolled, adaptation may lead to abnormal vessel wall remodeling with excessive neointimal hyperplasia with deposition of smooth muscle cells (SMC) and extracellular matrix (ECM), ultimately vein graft failure and clinical complications. Since1950autogenous vein has been widely used in patients with ischemic disease, but20-50%of vein grafts are ultimately failure because of its remodeling process leading to thrombosis, restenosis et al. Once restenosis occur after revascularization, vascular reconstructive surgery may be taken, which will lead to the formation of restenosis again. There is currently no effective treatment to reduce excessive thickening of the graft wall. The mechanism and prevention measures of graft restenosis have been the focus of clinical research.Numerous physiologic and molecular mechanisms of vein graft adaptation have been discovered. However, this complex process has yet to be characterized well, and we do not yet have the ability to therapeutically control graft wall thickening. Early restenosis is associated with acute thrombosis, intimal proliferation.Long-term restenosis is the pathematology alteration after operation. Vascular smooth muscle cells and extracellular matrix are the essential component of the neointima.The pathophysiology of vein graft failure involved a complex mechanism. Conventional pharmacotherapy had limited impact on intima proliferation of vein graft. Along with the development of gene engining and molecular biology, gene therapy may become the method to solve the problem. DNA/RNA can be encapsulated in nanoprecipitation or adsorbed into the surface of nanoprecipitation, which can be uptakeen into the cell to achieve the purpose of sustained and stable expression of the gene. Biodegradable PLGA nanoprecipitation play an important role in the study of nanoparticles because of sustained-release drug delivery systems.Arresten is the26kDa C-terminal globular non-collagenous(NC1) domain of the al chain of type IV collagen (al[IV]NCl)(Colorado et al.,2000), with molecular weight about26kD. It was initially isolated from human placental basement membrane. In recent years, some study showed that arresten could inhibit the endothelial cells’proliferation and migration, induce endothelial cells’apoptosis and also inhibit tumor growth and metastasis. In early study, arresten showed effective in the inhibition of proliferation of vascular smooth muscle cells and neovascularization. So arresten play an important roll in neintimal hyperplasia and restenosis after vscular recomstructive operation.OBJECTIVE Construction of eukaryotic expression vector of Arresten gene. Construction of Arresten gene loaded nanoparticles biodegradable composed of biodegradable and biocompatible materials PEG-PLGA, to construct rabbit autogenous vein transplantation model, and then the recombinant plasmid was locally transformed into the vein graft; and the influence on neointimal hyperplasia of vein graft was explored.METHODS(1) A synthetic design arresten sequences, according to building requirements at the5’end to add start codon ATG, and add the enhanced expression of kazak sequence GCCACC, remove the termination codon at the3’end. After loading sequence synthesis to pUC57carrier, enzyme digestion and sequencing were conduct. The conduction was inserted into pEGFP-Nl vector.The target gene sequence were affirmed correctly.(2) A modified nanoprecipitation method was established to formulate the target gene loaded PEG-PLGA nanoparticles. Accurately weighted (20mg) PEG-PLGA was dissolved in1mL DMSO containing200μg of plasmid DNA. The resulting clear solution was slowly (30mL/h) injected by a microsyringe pump into20mL magnetic ally stirring0.5%(w/v) Pluronic F127aqueous solution (600rpm) and agitated for5hours at room temperature. The DNA-loaded PEG-PLGA nanoparticles formed instantaneously.(3) Preparation of rabbit autogenous vein graft model, and the recombinant plasmids pEGFP-N1-arresten were locally transfected into vein graft. Detection of arresten expression in vein graft by RT-PCR. Intimal and medial hyperplasia degree was measured by the thickness, which was measured by computer after hematoxylin eosin stain and Masson stain. Smooth muscle alpha-actin (a-SMA)、Proliferating cell antigen (PCNA)、matrix metalloproteinase(MMP-2) and MMP-9of vein graft were analysised by immunohistochemical labeling.RESULTS(1) Sequence analysis showed that, the sequencing results for human Arresten cDNA was cloned in the same prostage sequence. Objective gene into the expression vector is correct. The Arresten gene fragment inserted into the vector between the coincidence. Indicated that arresten gene was cloned into the expression vector comprehensively, accurately to the correct expression of target protein. Plasmid pEGFP-Nl-arresten was successfully constructed.(2) In nano PEG-PLGA nanoparticles modified precipitation method has uniform spherical particles, an average of219.8±2.28nm with narrow size distribution, a negative Zeta potential in pH7.4-24.52±0.644mv. Using the nano modified precipitation method, the obtained PEG-PLGA nanoparticles exhibited greater loading efficiency (96.64±0.202%).(3) To establish the model of autogenous vein graft. RT-PCR results showed that the genome of arresten-transferred tissue contained fragment of arresten gene; about the thickness of the medial and intimal issue, arresten transfection group was less than the control group and blank group, and the difference was statistically significant (P<0.05). Vascular smooth muscle cells consisted in the hyperplasic intima majure; the number and ration of MMP-2、MMP-9and PCNA positive-stained in arresten transfection group was lower than that of the control group and blank group (P<0.05).CONCLUSIONS Using the nano modified precipitation method the obtained PEG-PLGA nanoparticles exhibited greater loading efficiency (>95%). Autogenous vein graft model, by end to end anastomosis between the jugular vein and the ipsilateral carotid artery could lead to restenosis of vein graft. The human Arresten gene transfection locally can inhibit intimal hyperplasia of vein graft, improve the degree of patency. They show a good prospect in clinical application in inhibition and cure of rstenosis after vascular transplantation.更多还原