【作者】 宁小晖；

【导师】 张澍；

【作者基本信息】 北京协和医学院 ， 内科学， 2012， 博士

【摘要】 目的缓慢型心律失常是临床上常见的心律失常类型,严重影响患者的生活质量和生命安全。20世纪50年代,心脏植入性电子起搏装置的发明,为该病的治疗带来了革命性的变化。然而,心脏电子起搏器终究只是一种缓解症状的治疗方法,在长期的使用过程中也出现了诸多缺陷。心脏起搏生理的基础研究方面的进展,为起搏功能障碍的治疗开辟了新的前景,生物起搏器的概念也应运而生。近十余年以来,生物起搏器的研究取得了巨大进展。目前,生物起搏器的体内实验主要通过外科开胸手术的途径进行移植,存在创伤大、定位效果差等缺点,因此移植途径是制约生物起搏器临床应用的重要因素之一。探索适宜的移植手段是促进生物起搏进入临床实验阶段所需要克服的重要问题。本课题以犬的骨髓间充质干细胞(Bone-Marrow Mesenchymal Stem Cells, BM-MSCs)作为基因转染的靶细胞,构建超极化激活环核苷酸门控阳离子通道(Hyperpolarization-activated Cyclic Nucleotide-gated channels2, HCN2)基因的重组慢病毒载体,转染BM-MSCs,研究和验证转染后BM-MSCs在体外的起搏功能。采用白行研究设计的经皮心肌内注射导管,通过介入方法将体外构建的生物起搏器移植到实验动物犬的体内,探索该方法的安全性和可行性。方法采用密度梯度离心法分离犬的BM-MSCs并进行培养。扩增目的基因mHCN2,构建重组慢病毒表达载体。重组质粒和慢病毒包装系统共同转染T293细胞进行慢病毒的包装。Real-time PCR法检测病毒滴度。Lenti-GFP-HCN2以不同感染复数(multiplication of infection, MOI)转染第3-4代BM-MSCs,确定最佳MOI。设置Lenti-GFP-HCN2转染的BM-MSCs为实验组,Lenti-GFP转染的BM-MSCs为阴性对照组,未转染的BM-MSCs为空白对照组。全细胞膜片钳检测各组BM-MSCs的起搏电流。分离培养新生乳鼠心肌细胞(neonatal rat’s cardiomyocytes, NRCs),将各组BM-MSCs和NRCs进行共培养,观察细胞搏动情况,并记录收缩频率。经颈动脉和股静脉途径分别进入犬的左、右心室,进行龙胆紫稀释液的多点注射,检验自行研究设计的经皮心肌内注射导管的操作性能。将16只健康成年杂种犬(20-25kg)随机分为实验组(注射0.6m1转染了Lenti-GFP-HCN2的MSCs,n=8),阴性对照组(注射0.6m1转染了Lenti-GFP的MSCs, n=4)和空白对照组(注射0.6ml生理盐水,n=4)。经颈动脉途径放入自行设计的注射导管,在左心室室间隔位置定点注射。术后埋植体外遥测的心电记录仪实时监测犬的心率和心律。移植术后2周,对实验动物犬进行双侧迷走神经刺激(S1S1周长100ms/60ms,电压3-5V,时程1min,间隔10min)。记录逸搏心率的频率,分析逸搏来源以及迷走神经刺激后出现逸搏心率的时间间隔。术后2周达实验终点,留取心脏组织标本,冰冻切片后一部分直接在荧光显微镜下观察荧光,一部分进行HE染色以及Cx43,GFP和CD68的免疫荧光染色。结果基因测序表明目的基因HCN2已经成功重组至慢病毒表达载体,重组质粒转染T293细胞后,western blot证实在蛋白水平上有HCN2的过表达。本实验中确定慢病毒感染犬BM-MSCs的最佳MOI为20,在转染后48h开始表达绿色荧光,5天达高峰。实验组BM-MSCs可记录到超极化激活的内向电流,而阴性对照组和空白组BM-MSCs在相同记录条件下未能检测到明显的超极化内向电流。实验组BM-MSCs有HCN2蛋白过表达,而阴性对照组和空白组BM-MSCs未见表达。BM-MSCs和NRCs共培养后第3天,实验组NRCs的细胞收缩频率较阴性对照和空白对照组显著提高(P<0.05)。经皮心肌内注射导管注射的有效性为91.6%,未出现严重并发症。移植术后各组的体外遥测心电记录仪显示室性早搏和室性心动过速的发生在各组间未见显著差异。双侧迷走神经刺激结果发现实验组的异搏心率主要来自注射腔左心室,频率为50.8±3.2bpm,而阴性对照组和空白组的异搏心率主要来自右心室,频率为42.2±6.1bpm(P<0.05)。免疫荧光检测显示介入方法移植到左心室的BM-MSCs在术后2周仍有存活,细胞之间有Cx43的表达和连接,HE染色和CD68的免疫荧光染色显示移植部位并没有显著炎症形成。结论mHCN2重组慢病毒(Lenti-GFP-HCN2)转染犬的BM-MSCs能在体外发挥起搏功能。自行研究设计的经皮心肌内注射导管可以用于在体移植转染的BM-MSCs,构建生物起搏器,该方法具有良好的有效性和安全性。利用该导管移植Lenti-GFP-HCN2转染的犬BM-MSCs可以在体内存活,并在移植部位重建起搏点,形成异位搏动。更多还原

【Abstract】 Objective:Bradyarrhythmia is a common arrhythmia type which severely affected the patient’s health. The invitation of cardiac implanted electronic devices in1950s is a marvel, and advances in this field have been impressive. However, it’s a relive therapy rather than cure, and important shortcomings exist. With the further development in cardiac pacing mechanism, the concept of biological pacemaker evolved, and the active investigation in the field of biological pacing is rapidly progressed. However, the in vivo animal studies of biological pacemaker are mainly performed through open thoracotomy, which cause large injury and had poor lacation controlling. Therefore, exploring optimal implantation approaches is very important for further clinic trails. This study used bone-marrow mesenchymal stem cells (BM-MSCs) as transfection target cells, and construct lentiviral expressing vectors carrying mouse Hyperpolarization-activated Cyclic Nucleotide-gated channels2(HCN2) gene. The in vitro pacing function of the transfected BM-MSCs was investigated. A self-designed percutaneous inject catheter was used to deliver the biological pacemaker into the left ventricular of canine, its feasibility and safety was explored. Meanwhile, the in vivo efficiency of the biological pacemaker was tested.Methods:Canine BM-MSCs was isolated by density gradient centrifugation and cultured. mHCN2gene fragment was amplified through PCR, then was connected with lentiviral vector. T293cells were con-transfected with lentiviral vector and packaging systems. The titers of lentiviral vectors were tested by real-time PCR. Gradient MOI of5,10,15,20,25,30was performed to determine the optimal MOI for the transfection of canine BM-MSCs. Set experiment group (Lenti-GFP-HCN2transfection), negative control group (Lenti-GFP transfection) and blank control group (with lentivirus transfection). Whole cell patch clamp tested the pacing current and western blot tested the HCN2protein expression of BM-MSCs in each group. NRCs were isolated and cultured, BM-MSCs was co-cultured with NRCs, the contraction frequency was observed and recorded. Catheter was inserted via carotid artery and femoral vein into left and right ventricle separately, diluted gentian violet was muitidot injected into the ventricle to testify the manipulation properties of the inject catheter.16health adult mongrel canine (20-25kg) were randomly divided into experiment group (0.6ml Lenti-GFP-HCN2 transfected MSCs was administrated, n=8), negative control group (0.6ml Lenti-GFP transfected MSCs was administrated, n=4), and blank control group (0.6ml saline was administrated, n=4). Inject catheter was inserted via carotid artery into left ventricle, septum inject was performed under the instruction of X-ray. Telemetering ECG recorder was implanted underneath the skin for heart rhythm monitoring.2weeks later, bilateral vague nerve stimulation was conducted (S1S1100ms/60ms, voltage3-5V, duration1min, interval10min). The escaping rhythm was recorded, its origin and frequency was analyzed. Heart tissue was obtained when end point was reached, frozen sections were sliced for GFP fluorescence, immunoflourescens stain and HE stain.Results:DNA sequencing demonstrated that mHCN2recombinated lentiviral vectors were constructed successfully. Western blot test confirmed that there was mHCN2protein expression in the T293cells, which was transfected with the mHCN2recombinated lentiviral vectors. The optimal lentivirus transfection MOI for canine BM-MSCs was20. Patch clamp showed the production of hyperpolarization-activated inward currents in experiment group while not in negative or blank control group. There was a high HCN2protein expression in experiment group other than negative and blank control groups.3days after co-culture, NRCs in experiment group revealed a significantly higher contraction frequency (P<0.05). The injection efficiency was calculated as91.6%, there was no severe complication associated with endomyocardial injection operation. Telemetering ECG recorder revealed premature ventricular contraction and ventricular tachycardia, and no significant differences were observed between three groups. Bilateral vague nerve stimulation showed that in experiment group, the escape rhythm mainly originated from left ventricle with an average frequency of50.8±3.2bpm, while the escape rhythm of negative and blank control groups from right ventricle, average frequency was42.2±6.1bpm (P<0.05). Histopathological examination proved that the injected BM-MSCs survived in vivo, there were Cx43connected between cells, HE stain and CD68immunoflourescens stain indicated no marked inflammation.Conclusions:In vitro transfection of canine BM-MSCs with Lenti-GFP-HCN2produced pacing current and expressed HCN2protein, indicating a potential of pacing function. The self-designed percutaneous endomyocardial inject catheter was proved to have highly-qualified efficiency and safety, it provided a better delivery for biological pacemaker study. Lenti-GFP-HCN2transfected canine BM-MSCs can be successfully delivered by this catheter, cells survived and reconstructed an ectopic pacing.更多还原