【作者】 侯永波；

【导师】 范全心；

【作者基本信息】 山东大学 ， 外科学， 2012， 博士

【摘要】 背景心脏是推动血液循环的动力器官,起着泵血的作用。心脏传导系统是由特殊分化的心肌细胞构成,产生并维持心脏正常的节律,保证心房、心室收缩和舒张的协调。心脏传导系统包括窦房结、结间束、房室结、希氏束、束支、蒲肯野纤维。心脏兴奋的正常传导依赖于整个传导系统的完整性。高度房室传导阻滞是心脏内外科多种疾病的常见并发症,比如缺血性心脏病及外科手术中房室结的损伤。1958年置入第一台全埋置式心脏起搏器,自此,人工心脏起搏成为高度房室传导阻滞的唯一治疗方法。近50年来,随着科学技术的不断进步,人工心脏起搏在工程技术方面都取得了长足进展,临床适应症不断拓宽,因此需要心脏起搏治疗的患者也越来越多。但是大量临床资料也已证实心脏起搏器的并发症甚多：1、与植入手术有关的并发症：心律失常、气胸和血气胸、囊袋出血、误穿锁骨下动脉和误置电极导线于左心室等。2、与组织损伤和炎症反应有关的并发症：囊袋伤口破裂、囊袋皮肤坏死和囊袋感染(严重者可致败血症)等。3、与电极导线有关的并发症：心肌穿孔、导线损坏、静脉血栓形成、心肌外肌肉收缩、输出阻滞和电极移位等。4、与起搏器有关的并发症：起搏器感知障碍、心动过速和起搏综合症等。5、起搏器电池寿命导致再次手术的可能、对于体格发育中的患者也有明显局限性。此外,安装起搏器的患者须避免进入有强电磁场的环境,不得接受电热治疗、微波治疗或中、高频治疗等,一些日用电器如电热毯、电动剃须刀的使用也受到限制,给患者的日常生活带来极大不便。由于起搏器存在的种种缺陷,人们期待一种更加安全可靠的替代疗法的出现。随着分子生物学的发展,人们试图发展生物起搏器。目前心脏生物起搏的研发策略主要是基因治疗,而基因治疗要成功地实施,必须具备三个关键因素：针对性的治疗基因；基因运送系统；基因表达调节系统。但是,相对于生物化学和分子生物学等学科对基因治疗技术的巨大促进作用,进展缓慢的基因运送系统已经严重制约着基因治疗的发展。虽然组织工程学在人工心脏瓣膜和血管的研究也都取得了重大进展,但对于房室传导阻滞的组织工程治疗研究,国外还处于起步阶段,国内也没有相关报道。2006年,美国波士顿儿童医院Choi YH等从胎鼠体内获得成肌细胞,并培养、接种到胶原质骨架结构,之后将其植入实验鼠房室沟内,试图用它来连接心房与心室间的信号传导。实验结果显示,这一培养组织可以很好地传导电脉冲信号,而且这种传导是永久性的。但同时实验也存在一些问题：1.由于实验鼠心脏太小,不能有效破坏房室结,影响了对经培养组织下传电信号的观察；2.由于心房和植入组织之间的局部传入阻滞,须额外低电流刺激才能观察电信号传导,而这个问题在大型动物模型中是可以避免的；3.虽然电脉冲信号能够下传,但培养组织并不能像正常房室结一样对信号进行延搁,所以导致了心房心室同时除极,影响了正常心脏功能。骨髓间充质干细胞(Mesenchymal stem cells MSCs)存在于骨髓基质,具有多向分化潜能。它可以向多种结缔组织和一部分来源于外胚层的组织分化,比如脂肪、软骨、骨、肌肉、真皮、神经等。骨髓间充质干细胞取材方便,在体外培养时具有黏附性,成纤维样生长,且表达特定的细胞表型,如CD29, CD44, CD90, CD105,而不表达CD34,CD45,因此易于鉴定和纯化。骨髓间充质干细胞可分化为多系细胞,来源比较充足,无免疫抗原特性,体外培养成活率较高,目前已成为心脏病细胞治疗最有潜力的种子细胞。缝隙连接又称通讯连接,包括代谢偶联和电偶联,广泛分布于动物组织细胞间,偶联细胞相互通讯。缝隙连接是紧密连接的特定区域,连接两个细胞膜,一个细胞膜上的结构被称为连接子,又叫半通道,是由六个亚单位—连接蛋白(connexin, Cx)分子构成的6聚体蛋白质。Cx普遍存在于心脏的工作细胞和传导系统的特化细胞中,只是在数量与分布形式上存在着差异,不同的心肌组织表达不同表型的Cx,形成具有特定的生物学特性的细胞间通道。在哺乳动物心脏上发现了不少于4种Cx的存在,心房肌细胞表达Cx43、Cx40、和Cx45,心室肌细胞表达Cx43和Cx45,各项研究表明Cx43是心室肌细胞间电流的主要导体。Maze手术的实践证明,适当设计成形的心房肌条可以用来充当心房—心室电信号传导的介质。因此,我们设计将右心耳翻转与右心室做吻合,以期望心房细胞和心室细胞间建立起缝隙连接,为增加连接面积,减少心电传导的电阻,我们在心房心室吻合面之间注入体外培养的骨髓间充质干细胞,以求达到心电更好的传导。第一部分：骨髓间充质干细胞的分离、体外培养、鉴定及标记研究目的：研究骨髓间充质干细胞的分离、体外的培养、鉴定并观察生物学活性,探讨4’,6二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole, DAPI)对于骨髓间充质干细胞标记效果。研究方法：1.骨髓间充质干细胞的分离、体外的培养、鉴定Histopaque密度梯度离心法从三月龄狗的髂骨骨髓血中分离出含有杂细胞的骨髓间充质干细胞,磷酸盐缓冲溶液(phosphate buffer solution PBS)洗两到三次,细胞种植到T75cm2塑料培养瓶中,培养瓶中为含有10%胎牛血清,青霉素(100μg/ml),链霉素(100μg/ml)的低糖DMEM培养液,在含5%CO2及95%湿度的37℃细胞培养箱中继续培养3天,培养细胞更换培养液,利用骨髓间充质干细胞贴壁生长的特性,黏附细胞可继续生长,未贴壁细胞则被抛弃。约10-15天培养后,黏附的细胞可呈集落式生长,达到70%-90%的融合。0.25%胰蛋白酶消化收集细胞,按1：2-3比例传代(104/cm2)。观察骨髓间充质干细胞形态,生长速度并绘制骨髓间充质干细胞的生长曲线。骨髓间充质干细胞的表面抗原检测：0.25%胰蛋白酶消化收集细胞,PBS洗三次,洗掉残留培养液以去除干扰,制备成106个/m1的细胞悬液100μ1,加入相应抗体20u1,避光、室温反应30min,1500rpm,室温离心10min。再次加入500μ1PBS重悬细胞,流式细胞仪检测细胞CD29, CD44, CD34。犬IgG-FITC作为同型对照。2.骨髓间充质干细胞体外DAPI标记在含有第三代骨髓间充质干细胞的塑料培养瓶中加入4’,6二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole, DAPI),使得DAPI最终浓度为50μg/ml,在含5%CO2及95%湿度的37℃细胞培养箱中孵育30min,移去培养液,PBS冲洗4次,尽量洗掉细胞外DAPI,荧光显微镜下观察骨髓间充质干细胞体外DAPI标记效果。结果：使用Histopaque密度梯度离心法结合贴壁筛选法可得到较高纯度的骨髓间充质干细胞,骨髓间充质干细胞通过体外培养扩增,培养5-7天可见MSCs呈集落聚集性生长,集落中央的细胞最为密集。呈现典型的梭形状,并呈旋窝状排列。生长曲线显示1-5天为潜伏期,7天以后则进入快速生长期,传代后的MSCs增值速度较原代培养细胞加快,第三代骨髓间充质干细胞已基本得到纯化。但是随着传代的增多,骨髓间充质干细胞增殖能力逐渐减弱,克隆形成率逐渐在降低。流式细胞仪细胞表面抗原检测CD34阴性,超过95%细胞表达CD29, CD44,符合骨髓间充质干细胞的特征。第三代骨髓间充质干细胞体外DAPI标记阳性率达到100%。结论：Histopaque密度梯度离心法结合贴壁筛选法可得到较高纯度的髓间充质干细胞,DAPI可有效标记骨髓间充质干细胞的胞核。第二部分：骨髓间充质干细胞辅助右心耳右心室吻合建立新的电传导通路的实验研究研究目的：翻转犬右心耳与右心室吻合建立新的电信号传导的通路,吻合面注入骨髓间充质干细胞(Mesenchymal stem cells MSCs)以达到减少电阻的目的,使心室易于起搏,并探讨其可能机制。研究方法：1岁龄雄性比格犬24只,体重12-15kg,随机分为三组,每组8只,分别为对照组,DMEM培养液组,MSCs组。制备右心耳翻转与右心室吻合动物模型,对照组仅给予右心耳翻转与右心室吻合；DMEM培养液组在吻合面注入0.3mlDMEM培养液;MSCs组在吻合面注入0.3mlDMEM培养液混悬DAPI标记的MSCs (1×108/ml).制备动物模型前,比格犬行心脏彩超检查,了解左心室射血分数(left ventricular ejection fraction LVEF),动物麻醉后左侧第四肋间进胸,给予右心耳翻转与右心室吻合处理,吻合面积约1cm2。8周后再次行心脏彩超检查对比术前、术后心脏功能有无明显变化；动物麻醉后循原切口进胸,切断右心耳,仅保留右心耳与右心室呈岛状连接,体外心脏起搏器刺激残留心耳,并记录右心室是否可有起搏及起搏电流的大小；留取吻合面周围心肌组织行缝隙连接蛋白43(connexin43)及心肌肌钙蛋白T (Cardiac Troponin T)免疫荧光双染色检测,了解MSCs在吻合面内向心肌细胞的转化效果。结果：右心耳翻转与右心室吻合动物模型制备前平均左室射血分数83.2±1.9%,8周后复查心脏彩超显示平均左室射血分数为82.5±2.2%,术前、术后LVEF无明显变化(P=0.09)。电子起搏器心室起搏实验中,对照组共有7只可成功起搏,平均起搏电流17.1±1.2mA, DMEM培养液组共有6只起搏,平均起搏电流17.8±1.2mA, MSCs组8只全部起搏,平均起搏电流10.8±0.7mA, MSCs组起搏电流较DMEM培养液组和对照组明显降低(P<0.01),DMEM培养液组和对照组起搏电流则无明显区别(P=0.25)。免疫荧光双染色可见右心房心室吻合面内仍有DAPI阳性的MSCs细胞,且该细胞同时表达心肌特异性的肌钙蛋白T(Cardiac Troponin T)和参与电机械偶联的缝隙连接蛋白43(connexin43)。结论：右心耳翻转与右心室吻合可建立电信号传导的有效介质,吻合面内注入的MSCs可转化成具有电信号传导功能的心肌细胞,并可明显减少电阻,降低最小起搏电流。更多还原

【Abstract】 BackgroundThe heart is the organ of dynamic to promote blood circulation, play the role of pumping. The cardiac conduction system is composed of a special differentiation of myocardial cells to produce and maintain normal heart rhythm, ensure the atrial and ventricular systolic and diastolic coordination. The cardiac conduction system including sinus node、internodal tract、atrioventricular node、His bundle、bundle and Purkinje fiber. The heart’s normal conduction is depended on the integrity of the entire conduction system.The advanced A-V block is a common complication of many cardiac diseases, such as ischemic heart disease and surgical damage of the atrioventricular node. The full embedded pacemaker was inserted in1958, since then, artificial cardiac pacing has become the only treatment for the advanced A-V block. The past50years, with the continuous progress of science and technology, the artificial heart pacing in engineering technology has made considerable progress, continue to broaden the clinical indications, therefore more and more patients in need of cardiac pacing. But a large number of clinical data has also been confirmed that many complications of cardiac pacemakers:1、Implantation-related complications:arrhythmias, pneumothorax and hemopneumothorax, pouch bleeding, mistakenly wear the subclavian artery and the misplacement of the electrode wire in the left ventricle.2、 Tissue damage and inflammation-related complications:capsular bag wound rupture, capsular skin necrosis and capsular bag infection (severe cases can lead to sepsis).3、 Electrode lead-related complications:cardiac perforation, wire damage, venous thrombosis, myocardial outside the muscle contraction, output block, and electrode displacement.4、Pacemaker-related complications:pacemaker perceived obstacles, tachycardia and pacemaker syndrome.5、Pacemaker battery life lead to the possibility of the reoperation, there are obvious limitations for the physical development of patients. In addition, patients with pacemaker should avoid entering into a strong electromagnetic field environment, may not accept the electrothermal therapy, microwave therapy, high frequency treatment, household electrical appliances such as electric blankets, electric shavers are also restricted which caused great inconvenience to the patient’s daily life.Because of the shortcomings of pacemaker, People look forward to the emergence of a more safe and reliable replacement therapy. With the development of molecular biology, People trying to develop biological pacemaker. Cardiac biological pacemaker Research and Development strategy is gene therapy, three key factors must have in order to ensure the gene therapy. Targeted therapeutic gene、gene transfer systems、regulation of gene expression systems. However, compare to the huge promoting role of gene therapy technology in biochemistry and molecular biology, the slow progress of gene delivery system has been seriously hampered the development of gene therapy. Tissue engineering research have made significant progress in artificial heart valves and blood vessels, but for the treatment of atrioventricular block in tissue engineering research is still in its infancy abroad, there is no relevant reports In the domestic.2006, Choi, YH who came from the United States Children’s Hospital Boston, obtained myoblasts from fetal mice and culture, inoculated into the skeleton structure of the collagen, and then implanted in mice atrioventricular sulcus, try to use it to connect between the atrial and ventricularsignal transduction. The experimental results show that this tissue culture can be a good conduction of electrical pulses, and this conductivity is permanent. But at the same time, there are some problems with the experiment:1、the heart of mice was too small, so the atrioventricular node could not be destroyed effectively, then affecting the observation of the transmission of electrical signals in the cultured tissue.2、because of the local incoming block Between the atrium and implantation of the local organization, an additional low current was wanted in order to observe the electrical signal conduction, and this problem can be avoided in large animal models.3、Although the electrical pulse signal could pass, but the cultured tissue did not have the function of signal delay compared with the normal atrioventricular junction, resulting in atrial ventricular depolarization at the same time, and affecting the normal cardiac function.Mesenchymal stem cells (MSCs) exist in bone marrow stroma with multiple differentiation potential. It can be a variety differentiation of connective tissue, and part comes from the ectoderm, such as fat, cartilage, bone, muscle, dermis, nerves, etc. Bone marrow mesenchymal stem cells obtained easily, in vitro adhesion, fibroblast-like growth, and the expression of specific cell phenotypes, such as for CD29, CD44, CD90, CD105, without expression of CD34, CD45, so it’s easy to be identified and purified. Bone marrow mesenchymal stem cells can differentiate into multi-lineage cells, adequate source, and no antigens features, survival rate is higher were cultured in vitro, so it has the potential to be a seed cells in heart cell therapy. Gap junctions, also known as the communication connections, including metabolic coupling and electrical coupling, are widely distributed in animal tissue cells, the coupling cells communicate with each other. Gap junctions is closely connected to a specific area, connecting the two cell membrane, a membrane structure is called a sub-connection, also known as semi-channel, is composed of six subunits-connexin (Cx) molecules consisting of six polymer protein. Cx commonly found in the specialized cells of the heart cells and the conduction system, but there are differences in the number and distribution, different myocardial tissue expression of different phenotypes of Cx, the channel with specific biological characteristics was formed between the cells. The existence of at least four kinds of Cx was found in mammalian heart, atrial myocytes expression of Cx43, connexin40, and Cx45, ventricular cells express Cx43and Cx45, Various studies have shown that Cx43is the main conductor of the current in ventricular myocytes.Maze surgery Practice has proved that properly designed shape of the atrial muscle can be used to act as atrial-ventricular electrical signal conduction medium. Therefore, we designed flipped the right atrial appendage anastomosised with right ventricular, the gap junctions between atrial cells and ventricular cells was expected to be established, in order to increase the connection area, reduce cardiac conduction resistance, in order to achieve better cardiac conduction, We injected MSCs which cultured in vitro into the surface of atrial ventricular anastomosis. Part one:The isolation、culture、amplification identification and labeling of MSCs in vitroObjective:To study the isolation, in vitro culture and amplifying method of MSCs and identifying the MSCs cultured by this method. Research the biological characteristics of MSCs and the result of4,6-diamidino-2-phenylindole labeling of MSCs.Method:1. The isolation、culture、amplification identification in vitroHistopaque density gradient centrifugation o isolated MSCs which contains miscellaneous cells from March age dog iliac bone marrow blood, washed twice to three times by phosphate buffer solution (PBS), the cells were seeded into the T75cm2plastic culture flasks, The low-glucose DMEM in the Culture flasks containing10%fetal bovine serum, penicillin (100μg/ml), streptomycin (100μg/ml), Cultured for3days in the37℃incubator with an atmosphere containing5%CO2and95%humidity, replaced with new medium, The adherent growth characteristics of MSCs was used, adherent cells continue to grow, not adherent cells were abandoned. About10to15days, the adhesion of cells can be presented to the colony growth, reaching70%-90%of the fusion.0.25%trypsin digestion and cells were collected,1:2-3ratio of passages (104/cm2). Observed the morphology and growth rate of MSCs, drew the growth curve of MSCs.MSCs surface antigen detection:The MSCs were digested by0.25%trypsin and then collected, PBS washed three times, washed residual medium in order to reduce interference, preparation of cell suspension100μl which the cell concentration of106/ml, adding the appropriate antibody20μl, incubated30min in the dark, at room temperature,1500rpm, room temperature, centrifuged for10min, to join500μl PBS resuspended cells again, Flow cytometry to detect the expression of cell phenotype CD29, CD44and CD34expression, The dog IgG-FITC as isotype control.2、MSCs labeled with DAPI in vitro 4、6-diamidino-2-phenylindole (DAPI) was added in the plastic culture flask which contained third generation of, making the final DAPI concentration of50μg/ml, incubated30min in the37℃incubator with an atmosphere containing5%CO2and95%humidity, removed the culture solution, PBS washed4times in order to remove the extracellular DAPI, The effect of MSCs labeled in vitro with DAPI was observed by fluorescence microscope.Result:High purity MSCs could be obtained by Histopaque density gradient centrifugation combined with adherent screening method, MSCs cultured in vitro and amplifyed, it can be seen were colony clusters of growth cultured for5-7days, colony of the central was most intensive. Showing the typical shape of the shuttle and showing a whirlpool-like arrangement. The growth curve showed1-5day as the incubation period,7days after then enter the platform growing season. The proliferation of MSCs after passage faster than primary cells, the third generation of MSCs has been purified basically. However, with the passage increased, the MSCs proliferation capacity weakened gradually, and the clone formation rate reduced gradually. Cell’s surface antigen detected by flow cytometry showing CD34negative, over95%of cells expressed CD29, CD44, consistent with the biological characteristics of MSCs.Conclusion:High purity MSCs could be obtained by Histopaque density gradient centrifugation combined with adherent screening method, DAPI could label the nucleus of MSCs effectively. Part two:Establishing a new electrical conduction pathway by anastomosis of the right auricle and right ventricle assisted by mesenchymal stem cells in a canine modelObjective:The right atrial appendage and right ventricle was anastomosed in order to establish a new electrical conduction pathway, for the purpose of reducing resistance and ventricular pacing easily, MSCs were injected into the anastomosis surface, at the same time to explore its possible mechanism.Method:1year old male beagle dogs24and weight12-15kg, were randomly divided into three groups (n=8), respectively, for the control group, DMEM medium group, MSCs group, Before preparation of animal models Beagles accepted echocardiography examination In order to obtain the left ventricular ejection fraction (LVEF) and other parameters. The animals were anesthetized and the heart was accessed through an anterior right-sided thoracotomy at the fourth intercostal space, the right atrial appendage and right ventricle was anastomosed gently, An area of about1cm2.The control group only received the right atrial appendage turned downward anastomosis with right ventricular,0.3mlDMEM medium was injected into the anastomosis surface in DMEM supplemented group, the anastomosis surface of MSCs group was injected0.3ml DMEM suspension of MSCs labeled with DAPI (1×108/ml). Echocardiography was performed under light anesthesia in all animals to assess the cardiac function before the operation and8weeks after the anastomosis. We took the original surgical incision into the chest, cut off the right atrium, remaing the only connection of right atrial appendage and right ventricular, we stimulated epicardial and endocardial surfaces of the right atrial appendage near the site of anastomosis to verify existence of a secondary electrical pathway by pacemaker. Myocardial tissue surrounding anastomosis surface was obtained to do double immunofluorescence staining of gap junction protein43(connexin43) and cardiac troponin T, at the same time to detect the conversion of MSCs.Result:The average of left ventricular ejection fraction (LVEF) was83.2±1.9 before surgical procedure, the review of echocardiography eight weeks after the surgical procedure showed a mean LVEF82.5±2.2%, there was no significant change of LVEF Preoperative and postoperative (P=0.09). Seven ventricular were paced successfully by electronic pacemaker in Control group with average pacing current17.1±1.2mA; six ventricular were paced successfully by electronic pacemaker in DMEM group with average pacing current17.8±1.2mA; total eight ventricular in MSCs group were paced successfully and the average pacing current was10.8±0.7mA. The pacemaker current of the MSCs group decreased significantly (P<0.01) compared with DMEM and control groups, there was no significant difference (P=0.25) between DMEM group and control group. Double immunofluorescence staining showed the DAPI-positive MSCs between right atrium and ventricle anastomosis surface, the cells expressed cardiac-specific troponin I and gap junction protein43(connexin43) which involved in electromechanical coupling.Conclusion:Electrical signal conduction medium could be created effectively through the anastomosis of right atrial appendage and right ventricular, MSCs injected in the anastomosis surface can convert into myocardial cells with the function of electrical signal conduction. MSCs could reduce resistance and minimum pacemaker current significantly.更多还原