【作者】 张涛；

【导师】 邹承伟；

【作者基本信息】 山东大学 ， 外科学， 2014， 博士

【摘要】 研究背景人工电子心脏起搏器(cardiac pacemaker)作为治疗某些心律失常(主要是缓慢性心律失常)时首选的治疗仪器,是通过一种植入于人体内的电子脉冲发生器发放电脉冲,进而使与电极所接触的心肌细胞产生有节律的冲动,使心脏在此节律下有规律的激动和收缩,从而达到治疗的目的。自从1958年Rune Elmqvist成功将制造出的第一台人工心脏起搏器植入到患者体内至今,在缓慢型心律失常(如病窦综合征和重度房室传导阻滞等)治疗方面,人工起搏器已经被公认为首选的治疗方法。不过在人们的应用过程中,人工起搏器自身存在的一些缺陷及问题不断暴露出来,其中比较严重的包括：电极易于发生断裂、外来植入物可能导致感染、静脉内血栓形成而导致的栓塞、电池维持时间有限需重新进行手术更换等。除此之外,对于特殊患者特别是儿童而言,随着患儿的不断成长和发育,早先所安装的起搏器会逐渐无法满足机体的需要,因此大多需要进行重新更换,这都限制了它在某些特定患者中的应用。近些年随着分子生物学的发展,为了克服人工起搏器所存在的以上问题,有学者提出了生物起搏器的概念并被寄予厚望。目前,构建生物起搏器的方法主要包括细胞治疗和基因治疗两大方向,但是因为细胞治疗存在的一些问题如：伦理问题、成瘤问题、免疫排斥问题以及效果欠佳等,限制了其在这一领域的应用。而通过基因治疗构建心脏生物起搏器却越来越引起相关研究者的重视,现在基因治疗主要包括以下三种方式：(1)通过过度表达p2肾上腺素受体基因来增加心房的电活动；(2)过度表达或移植人工制造的工程化起搏电流(HCN2)基因来构建起搏细胞；(3)通过基因技术抑制内向整流钾电流(Ikl),引起心肌细胞钾电流的平衡发生变化,进而使无起搏活性的心肌细胞产生自律性。心肌是由心肌细胞构成的一种肌肉组织,广义上说,除了包括我们熟悉的具有收缩功能的工作细胞心房肌和心室肌以外,还包括无收缩功能的窦房结、结间束、房室交界部、房室束(即希斯束)和浦肯野纤维等5种特殊分化了的心肌细胞。后5种心肌细胞没有收缩功能,但是具有自律性和传导性,它们共同组成心脏的起搏和传导系统,是心脏能够进行自律性活动的结构基础；前两种心肌细胞具有收缩性,是心脏舒缩活动的功能基础。早期研究发现,成人心室肌细胞有潜在的起搏活性；另外在心肌Ikl中KCNJ2基因所编码的通道蛋白Kir2.1占80%左右,它可以使成人心室肌细胞的静息膜电位保持在负电位水平,而且它高度表达于没有自律性的心房肌和心室肌中,而在窦房结等具备自律性的细胞中表达却相对罕见或缺乏。因此有学者认为,由于受到Ikl的影响,心室肌的起搏活性才会被抑制。以上观点在后期的实验中得到了证实,其中在Silva J等的研究中发现,当对心室肌细胞的Ikl电流的抑制率达到81%以后,其潜在的起搏活性就会显现出来,出现自发的动作电位,而且其自发的搏动频率会随着Ik1电流抑制率的增加而相应的提高。而Miake等则应用基因工程构造出编码没有功能、非活性的IK1通道的Kir2.1AAA基因,通过负显性法来抑制心室肌细胞的Ikl电流。研究中他们发现在没有对豚鼠心脏正常兴奋性进行负性干预的前提下,成功的诱导出了起源于豚鼠心室肌细胞的自发起搏活动,而且经过检测此起搏活动的频率比窦房结的正常频率还要快,被认为是世界上首例成功构建的生物心脏起搏器。另外,有研究者通过动物实验还发现,如果将心肌细胞中KCNJ2基因的表达完全阻断,会导致多种心律失常的出现。Chan YC等进一步的研究则发现,在生物起搏器的基因治疗中抑制Kir2.1蛋白表达与过表达HCN4基因有协同作用,可以共同提高心肌细胞的自律性。近期,位于美国的Cedars-Sinai心脏研究所的KapoorN等通过向普通心肌细胞中注入单个Tbx18基因对其进行重新编程,将心肌细胞改造成专业化很高的“生物起搏器”的实验获得了成功,另外他们还发现改造后的心肌细胞所产生的电活动,可以规律的传导至全部心肌细胞,引起心脏出现有节奏的舒缩,证实了通过植入单个基因即可以将心肌细胞改造成专门的起搏细胞,并且通过心室的这个起搏点能够引起心脏出现规律的肌肉活动。这一事实进一步为通过提高局部心室肌细胞自律性来在体构建生物起搏器提供了依据。RNA干扰(RNA interference, RNAi)技术是目前比较成熟的一种基因干预技术,是由双链RNA(dsRNA)诱导的同源mRNA降解的过程[14,15]。能够达到在转录水平、转录后水平和翻译水平上特异性阻断基因表达的效果。目前人们已经掌握了根据RNA干扰的原理人工合成siRNAs (small interference RNA)的技术,为通过使用能表达siRNAs的载体在体外或体内诱导哺乳动物的细胞产生特异的基因沉默提供了技术支持。慢病毒载体(Lentiviral vector,LVs)是在人免疫缺陷病毒1型(HIV-1)基础上改造而成的,能利用逆转录酶和整合酶,将自身的RNA转变为DNA整合到宿主细胞的染色体中,从而使目的基因在宿主细胞中长期而稳定的表达。因为其具有宿主范围广,所能容纳的外源性基因片段大,安全性高等优点,目前在基因治疗中的应用越来越广泛。前期本课题组已经获得了干扰KCNJ2基因mRNA的特异位点(+361～+379碱基),并且通过应用设计出的特异干扰序列,在细胞水平证实了将RNA干扰基因导入心室肌细胞,使其产生干扰效应,特异性得使KCNJ2基因沉默,抑制内向整流钾电流(Ik1),进而诱导处于静息状态的心室肌细胞产生生物起搏活性的可行性。在本课题中,我们将在动物体内通过干扰KCNJ2基因来抑制模型大鼠心肌的内向整流钾电流(Ik1),探讨是否可以提高模型大鼠的心室率。在实验中我们通过建立大鼠Ⅲ度房室传导阻滞的模型,消除窦性起搏对心室肌细胞自律性的掩盖；进而应用慢病毒载体介导的RNAi技术,通过干扰KCNJ2基因从而抑制Kir2.1蛋白,达到抑制内向整流钾电流(Ik1)通道的目的,观察模型大鼠心室率的变化情况来探讨以上假设的可行性。希望可以为生物起搏器的体内研究提供一些有价值的试验依据。第一部分携带有特异性干扰片段的慢病毒载体的构建研究目的制备含有针对大鼠心肌细胞KCNJ2基因nRNA的特异性shRNA的慢病毒表达载体。研究方法1、将前期验证的对KCNJ2基因mRNA上沉默效果最明显的干扰片断以MluI,ClaI为插入位点,插入到由H1启动子调控以及有GFP表达的慢病毒载体中；并通过MluI,ClaI双酶切技术进行鉴定。2、在脂质体的介导下将混合的慢病毒载体包装质粒和包含KCNJ2基因干扰片段的重组慢病毒载体共转染293T细胞,包装成病毒,72小时后收集上清,应用逐孔稀释滴度测定法测定病毒滴度。研究结果通过双酶切电泳证实所设计的KCNJ2基因shRNA正确插入到了慢病毒载体中,DNA测序证实插入的序列正确；293T细胞成功包装重组慢病毒载体；收集的细胞培养上清液中,病毒的滴度为1.07×109TU/ml。结论制备的慢病毒载体构建成功,同时获得的病毒滴度较高,完全满足后续在体实验的要求。第二部分在大鼠III度房室传导阻滞模型中抑制心室肌KCNJ2基因的表达对心室率的影响研究目的1、建立稳定的大鼠III度房室传导阻滞的模型。2、确定慢病毒载体转染大鼠的最佳滴度。3、明确慢病毒载体转染大鼠III度房室传导阻滞模型后心室率的变化情况。4、明确慢病毒载体转染心肌细胞后KCNJ2基因mRNA和Kir2.1蛋白表达的变化及其于大鼠心室率的关系。研究方法1、采用定点注射70%乙醇的方法破坏大鼠的房室结,使大鼠发生III度房室传导阻滞。注射乙醇约25u1,观察心电图变化,出现III度房室传导阻滞的大鼠观察30分钟,待稳定后关胸,72小时后检测确定建模是否成功。2、取正常大鼠给予不同滴度的空白慢病毒载体30ul进行心肌注射,分别取注射后第4天、7天、10天处死取材,迅速制作冰冻切片,在免疫荧光显微镜下进行检测,根据各不同时段切片中的绿色荧光蛋白的亮度,确定转染效率与病毒滴度的关系,用转染效率最高的滴度进行模型大鼠的注射。3、将建立的模型大鼠分为对照组、空载体组、病毒干预组三组；其中对照组只进行转染的手术操作但不进行相应的病毒转染；另两组则分别转染慢病毒空载体及携带有相应shRNA的慢病毒载体。转染后分别于不同时间点通过心电图检测各组大鼠心室率的变化情况。4、分别对各组所取得的心肌组织进行相应处理,通过实时荧光定量RT-PCR检测KCNJ2基因的表达情况；应用Western-blot及免疫组化检测心肌组织中kir2.1蛋白的表达情况。研究结果1、建立了持久、稳定的III度房室传导阻滞大鼠模型,满足本实验的要求。2、慢病毒的最佳转染滴度为109TU/ml,转染7天后转染效率变稳定。3、本研究发现病毒干预组中有54.5%的大鼠心室率由156±6次／分钟提高到218±10次／分钟,差异性显著(p<0.01)；进一步实验证实心室率的提高与KCNJ2基因、Kir2.1蛋白的表达呈负相关；是由慢病毒转染的基因沉默引起的,且在转染后14天,当KCNJ2基因与Kir2.1蛋白抑制率分别为77%、55%左右时,大鼠的心室率达最快。因此证实了通过抑制心肌的KCNJ2基因及Kir2.1蛋白的表达,进而抑制内向整流钾电流(Ikl),能够引起III度房室传导阻滞模型大鼠心室肌细胞自律性的提高。结论在动物体内通过抑制KCNJ2基因来抑制模型大鼠心肌细胞的内向整流钾电流(Ikl)来提高大鼠的心室率是可行的。更多还原

【Abstract】 BACKGROUNDArtificial electronic cardiac pacemaker is an electronic instrument implanting into the inside of the body.The electrical pulse produced by pulse generator which stimulating the adjacent myocardial cells, it make the heart exciting and contraction, so as to achieve treatment for some arrhythmia (mainly bradycardiac arrhythmia). Since the first cardiac pacemaker implantation in the human body in1958, it has gradually become the preferred treatment of slow arrhythmia such as sick sinus syndrome and severe atrioventricular block. With the pacemaker continuous improvement, its clinical indications is extended continuously, which gradually began to apply to the tachyarrhythmia and non electrical diseases, such as preventing paroxysmal atrial tachyarrhythmia, carotid sinus syncope, refractory congestive heart failure by biventricular pacing, etc. But in the process of its application some defects and problems have become obvious, such as limited battery life, infections, reoperations and venous thromoembolus,etc. Furthermore, they are not suitable for patients that apt to infection or being too young. With the progress of molecular biology in recent years, people try to take advantage of life science and technology research to develop a biological pacemaker to replace electronic pacemaker in order to overcome the above problems. The new technigue could repair or replace the native cardic pacemaker and the damaged conduction tissue to restore the heart pacemaker and conduction function.At present, there are two major strategies in developing biological pacemaker:gene therapy and cell therapy. Cell therapy is definited as the method of applying stem cells (embryonic stem cells and mesenchymal stem cells) and sinus node cells. But there are lots of unsolved problems such as:immunne rejection, oriented differation, tumorigenicity and ethinic issue, etc. Developing biological pacemaker by gene therapy is rooted on three strategies:(1) Over-expressing the neurohormone receptors to increase the atria electric activity.(2)Over-expressing the HCN2in diastolic phase.(3) Suppressing the inward-rectifier potassium current(Ikl) to break the balance of the potassium currenct inside the ventricular cells, which then can obtain the capability of automatic rhythmicity.Myocardium is a muscle tissue that is composed of myocardial cells.The generalized myocardial cells not only included atrial and ventricular muscle but also the S-A node,internodal bundle, atrioventricular bundle, atrioventricular junction area and Purkinje fibers. The previously study demonstrates that adult myocardial cells possess the latent of pacing ability, which it is inhibited by IK1. With the aid of powerful inward rectifier properties holding the rest potential at negative level, IK1then is able to inhibit the myocyte spontaneous depolarization. IK1potassium is coded by gene KCNJ2, abundant in atrial and ventricular myocytes while sinus node cell is lack of this kind of channel. It is then assumed that ventricular myocyte can be changed to pacemaker cell if the IK1is inhibited. Silva J and Rudy Y found that after suppression Ikl by81%, the ventricular myocytes will generate a spontaneous action; and the more on IK1is inhibated, the higher the pacing rates of myocytes is. In2002, Miake reported a biological pacemaker created by dominant-negative therapy. They got spontaneous ventricular rhythm which was more rapid than that caused by the native sinus pacemaker. The beating rates of myocytes can also respond positively to β receptors agonist, creating rudiment for the future improvment of biological pacemaking. However, in the phenotype of completely lack of IK1, the mice bears Anderson’s syndroms, such as QT-prolongatioin, periodic paralysis, skeletal and craniofacial abnormalities.RNA interference (RNAi) is a phenomenon of gene silencing at the lever of post transcription resulted from the degradation of mRNA reduced by double strands RNA.RNAi was firstly founded in Caenorhabditis elegans. Recently,with the deepening of the research on RNAi, people have been able to induce specific gene silencing of mammalian cells by way of artificial siRNA (small interference RNA) synthesized according to the principle of RNAi in vitro or expressed through vector in vivo.Lentiviral vector was transformated on the basis of HIV-1. Lentiviral vector could mediate exogenous gene expression in host cell sustained.It has many advantages, such as wide host range, more stable, accommodate bigger exogenous gene fragment, high security and so on. Thus, the lentiviral vector has become the most used vector in gene therapy.In our previous studies, we has screened out the most significant suppressing sites on KCNJ2gene mRNA,namely+361-+379bp;and found it is feasible to increasethe heart rate by using RNAi to knock down the KCNJ2targeting gene in vitro.In this study we want to use RNA interference to knock down the KCNJ2targeting gene in vivo.We observed the effect of siRNAs transfection to inhibit the KCNJ2gene expression on ventricular rate in the Ⅲ°AVB rat model, so as to provide a new effective idea and approach for the study of biological pacemaker.Part1. Construction of lentiviral vector with the shRNA effect to KCNJ2ObjectiveTo construct specific lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA.MethodAccording to our previous research, the double-stranded DNA oligo which can cause most RNAi effect to KCNJ2was made. The small hairpin RNA(shRNA) sequences were annealed and linked with linearized into the lentiviral vector. Moreover, the recombinant lentivirus was harvested from293T cells when it cotransfected with lentiviral packing materials into them after72h. The virus particles were collected. Virus titer was determined by hole dilution method.ResultThe shRNA sequences were successfully inserted into lentiviral vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of KCNJ2gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of1.07x109TU/ml.conclusionThe lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA was successfully constructed.Part2. Experimental study on inhibition of ventricular Ikl by RNA interference targeting the KCNJ2gene in three degree atrioventricular block ratObjective1.To establish a stable rat three degree atrioventricular block(Ⅲ°AVB) model;2.To confirm the best virus titer to lentiviral vector infection in rat;3.To observe the variation of the heart rate(HR)after the lentiviral vector infection;4.To observe the alteration of the expression of KCNJ2gene and the Kir2.1protein after the lentiviral vector infection.Method1.A50ul needle was used to inject the solutions into the myocardium toward the nodal tissue,When the insertion of the needle resulted in momentary complete AV block,25ul of70%ethanol were injected. Hearts were reinjected with ethanol if the heart block resolved after30min. After72hours, the Ⅲ°AVB was defined as stable;2.The rats were infected by lentiviral vector of different virus titer; we drawn material at different time and made frozen sections. the infection rate was detected by immunofluoresc-ence microscopy, then confirmed the best virus titer to lentiviral vector infection in rat;3.The Ⅲ°AVB rat models were divided into three groups (1) Interference group: infected lentiviral vector with shRNA;(2)Negative control group:infected negative lentiviral vector;(3) Controll group:no treatment.The HR was analyzed with electrical cardiogram;4.The levels of target genes mRNA and protein were analyzed with the real time quantitative (RT-PCR), Western blot, Immunity histochemistry.Result1.the Ⅲ°AVB rat model was stable;2.The best virus titer to lentiviral vector infection in rat was1×10TU/ml;3.The HR of rat was increase after the lentiviral vector infection;The expression of KCNJ2gene and Kir2.1protein were suppressed after the lentiviral vector infection.ConclusionIt is feasible to increase the HR by using RNAi to knock down the KCNJ2targeting gene in vivo.更多还原