【作者】 童敏；

【导师】 杨向军；

【作者基本信息】 苏州大学 ， 心血管内科学， 2010， 博士

【摘要】 目的:研究超极化激活环核苷酸门控阳离子通道基因亚型4(HCN4)改造的大鼠骨髓间充质干细胞(MSCs)与心室肌细胞共培养,构建具有自律性的心脏生物起搏器模型的可行性;并研究MSC中过表达连接蛋白45(CX45)对以上生物起搏器模型自律性的影响。方法:1)体外分离培养新生大鼠的心室肌细胞,鉴定纯度及活力;并通过膜片钳方法记录心肌细胞的自发性动作电位。2)基因克隆的方法构建包含HCN4基因的穿梭质粒pCDH1-GFP-HCN4,并通过四质粒系统包装成慢病毒颗粒,转染大鼠的MSCs,构建成HCN4+MSCs;将仅转染有GFP基因的慢病毒颗粒的MSCs设为HCN4-MSCs。通过RT-PCR,以及荧光观察鉴定HCN4基因在MSCs中的表达;电压钳记录阳性转染细胞的起搏电流If。3)将HCN4+MSCs与乳鼠心室肌细胞共培养构建心脏生物起搏器模型,同时将HCN4-MSCs与心肌细胞共培养设为“HCN4-MSCs对照组”。为了监测生物起搏器的自律性,初步分析自律性变化的原因,计数心室肌细胞自发搏动频率、记录自发性动作电位;并通过免疫荧光标记等方法鉴定共培养的两类细胞间连接蛋白43(CX43)的表达;加入缝隙连接阻断剂甘珀酸验证缝隙连接在模型中的价值。4)为了构建CX45基因稳定表达的MSCs细胞株,首先通过基因克隆的手段构建含有CX45基因的表达质粒pDsRED2-N1-RFP/Gja7,通过脂质体2000的介导将pDsRED2-N1-RFP/Gja7转染MSCs;G418筛选获得稳定转染的细胞株CX45+MSCs;同时将pDsRED2-N1-RFP空质粒转染的MSCs设为CX45-MSCs。为了鉴定MSCs中CX45基因的转录,进行了RT-PCR试验;CX43与CX45单克隆抗体免疫双标MSCs,鉴定CX45+MSCs中连接蛋白类型的变化。5)将CX45+MSCs作为生物起搏的载体细胞,转染HCN4基因,构建HCN4+CX45+MSCs;将HCN4+CX45+MSCs与乳鼠心室肌细胞共培养重新构建心脏生物起搏器;同时将CX45-MSCs中转染HCN4基因,与心肌细胞共培养后构建的生物起搏器模型设为“HCN4+CX45-MSCs对照组”。通过记录共培养体系中乳鼠心室肌细胞自发搏动频率以及动作电位特征的改变,监测生物起搏器的自律性;免疫双标鉴定共培养的两类细胞间CX43和CX45的表达;了解缝隙连接阻断剂甘珀酸对该生物起搏器模型自律性的影响。结果:1)该方案分离培养的新生大鼠心室肌细胞消化瞬时的活细胞率为80%;培养至第5天时85%左右的心肌细胞都有自发性搏动,搏动频率80次/min左右;培养至第9天后心肌细胞纯度开始大大降低;培养5天心肌细胞的自发性动作电位阈电位为-60mV,频率84±12次/分,节律不规则。2)通过酶切鉴定以及基因测序证实HCN4基因成功构建到pCDH1-MCS1- EF1-copGFP质粒中,形成包含目的基因的穿梭质粒pCDH1-GFP-HCN4;通过穿梭质粒的介导成功将HCN4基因包装成慢病毒载体lentiv-GFP/HCN4,real-time PCR测定lentiv-GFP/HCN4滴度为3×108TU/mL。通过慢病毒载体介导HCN4基因转染MSCs,5天后,荧光显微镜下观察转染阳性率约70%;RT-PCR证实HCN4+MSCs中有HCN4 mRNA的表达;膜片钳记录到HCN4+MSCs上有时间和电压依赖性的超极化激活的内向电流,该电流的激活阈电压为-70mV,对低浓度CS+敏感,符合If的特征。至此说明,MSCs上已经成功表达有起搏离子通道HCN4。3)将HCN4+MSCs与3d心肌细胞共培养2-4d后,发现实验组心肌细胞的自发搏动频率明显快于“HCN4-MSCs对照组”中的心肌细胞自发搏动频率。膜片钳结果显示心肌细胞的自发动作电位频率显著提高(129±11次/分vs. 82±8次/分,P<0.05,n=5);动作电位频率变规则;动作电位最大舒张电位(MDP)绝对值降低(-66±6mV vs.-87±4mV,P<0.05);而实验组与对照组的阈电位均为-60mV左右。免疫荧光标记证实共培养的两类细胞相接触部位的膜上有CX43的表达;加入缝隙连接阻断剂甘珀酸后,观察实验组心肌细胞的自发搏动频率明显降低,而且变的不规则。综上,HCN4基因改造的MSCs与心室肌细胞共培养,成功构建成具有自律性的心脏生物起搏器模型;缝隙连接在生物起搏器模型中具有重要价值。4) PCR产物琼脂糖凝胶电泳以及基因测序证实了CX45(Gja7)基因成功插入表达载体pDsRED2-N1-RFP中,形成包含CX45基因的质粒pDsRED2-N1-RFP/ Gja7。脂质体2000介导pDsRED2-N1-RFP/CX45瞬时转染MSCs 48小时后,荧光显微镜下观察发现阳性率达到60%;经过50μg/ml G418筛选获得稳定表达CX45基因的MSCs细胞株CX45+MSCs。RT-PCT证实CX45+MSCs上有CX45 mRNA的表达;免疫双标检测到CX45+MSCs细胞膜上同时表达有CX45和CX43。至此说明,CX45基因已经改变了MSCs细胞膜上连接蛋白分布,形成了一种细胞膜上同时高表达CX45和CX43的细胞株。5)将HCN4基因转染CX45+MSCs,与心肌细胞共培养,构建新的生物起搏器模型中心肌细胞的自发动作电位频率显著高于“HCN4+CX45-MSCs对照组”(147±9次/分vs. 123±8次/分,P<0.05);动作电位4相自动去极化速度(VDD)也明显提高(0.065±0.016V/s vs. 0.021±0.009 V/s,P<0.05)。免疫双标证实心肌细胞与CX45+MSCs相接触部位有CX43与CX45的表达。加入甘珀酸后,实验组和“HCN4+CX45-MSCs”心肌细胞的自发搏动频率均明显降低,实验组尤其明显。综上,MSCs细胞膜上CX45的高表达,使得生物起搏器模型的自律性提高。结论:HCN4基因改造的MSCs与心室肌细胞共培养,成功构建成具有自律性的心脏生物起搏器模型;MSCs细胞膜上CX45的高表达,使得以上生物起搏器模型的自律性提高。更多还原

【Abstract】 Objective: To recreating a cardiac biological pacemaker based on bone marrow-derived mesenchymal stem cells (MSCs) which transduced with hyperpolarization- activated cyclic nucleotide-gated cation channel gene subtype 4 (HCN4); And to study the impact of changed connexin subtype by overexpression with connexin45 in MSCs on automaticity of biological pacemaker.Methods:1) Neonatal rat ventricular myocytes were dissociated and cultivated as previously described; The purity and viability of myocytes were tested; Then spontaneous action potentials of myocardial cells was recorded by the patch-clamp technique.2) The shuttle plasmid containing HCN4 gene (pCDH1-GFP-HCN4) was built by gene cloning; pCDH1-GFP-HCN4 was packaged into lentiviral particles; Lentiviral particles containing HCN4 was transfected into rat MSCs; Then HCN4+MSCs had been constructed; MSCs transfected with lentiviral particles only containing GFP gene was defined as HCN4-MSCs; HCN4 expression in MSCs was conducted by RT-PCR, and observation of GFP fluorescence under fluorescence microscope. If current in HCN4+MSCs was recorded by Voltage-clamp.3) HCN4+MSCs and neonatal rat ventricular myocytes were cocultured to build biological pacemaker model; Where HCN4-MSCs and myocytes cocultured was defined as " HCN4-MSCs control group"; To evaluate automaticity of biological pacemaker, spontaneously beat frequency of myocytes was counted, and spontaneous action potentials of myocytes was record by current patch-clamp. Immunostaining of CX43 between myocytes and MSCs was conducted with CX43 Monoclonal antibody. Carbenoxolone (200 microM), a gap junction channel blocker, was added into the model to determine the impact of gap junction channel on biological pacemaker model.4) And to construct MSCs stable overexpression with CX45, pDsRED2-N1-RFP/ Gja7 containing CX45 gene(named as Gja7, as well) was built by means of gene cloning, then transfected into MSCs with the help of Liposomes 2000. A stable cell line CX45+MSCs was acquired with the stress of G418; MSCs transfected with empty plasmid pDsRED2-N1-RFP were defined as CX45-MSCs. To test the transcription of CX45 gene in MSCs,RT-PCR was carried out; To verify the connexins in MSCs had been changed, double immunostaining was conducted with a cocktail of monoclonal antibody CX43 and CX45 in CX45+MSCs.5) To recreate a biological pacemaker based on changed connexins in MSCs, CX45+MSCs was transduced with HCN4 gene. As a result, HCN4+CX45+MSCs was constructed. Then HCN4+CX45+MSCs were cocultured with neonatal rat ventricular myocytes to recreate a biological pacemaker; As a control, CX45-MSCs transduced with HCN4 were cocultured with myocytes simultaneously. To evaluate automaticity of biological pacemaker as described above. Double immunostaining of CX43 and CX45 between heterologous cell pairs was conducted with a cocktail of CX43 and CX45 monoclonal antibody. Carbenoxolone was added into the model to determine the impact of gap junction channel on transformed biological pacemaker model .Results:1) The survival rate of neonatal rat ventricular myocytes was about 80% just after the digestion. 85% myocytes beating spontaneously at the frequency of about 80 beating per minutes(bpm) in the fifth day. The purity of myocytes was greatly reduced after 9 days. Spontaneous action potentials outbreak with the mean rate of 84±12 bpm and a irregular cycle in neonatal rat ventricular myocytes. Threshold potential for action potentials of myocytes was about-60mV. 2) Restriction enzyme digestion and gene sequencing confirmed HCN4 gene had been successfully built into the shuttle plasmid pCDH1-GFP-HCN4. Titer of lentiviral vector lentiv-GFP/HCN4 packaged with pCDH1-GFP-HCN4 was 3×108TU/mL. The transfection efficiency for HCN4 by the means of lentiviral vector was 70% after 5 days. RT-PCR confirmed the transcription of HCN4 gene in MSCs. High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4mmol/L CsCl was detected in HCN4+MSCs, confirming that HCN4 acted as If channels in MSCs.3) The effects of genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Counted spontaneously beating frequency in myocytes coclutured with HCN4+MSCs was greatly faster than in which myocytes coclutured with HCN4-MSCs. Recorded action potential in myocytes demonstrated that myocytes coclutured with HCN4+MSCs was characterized with a more regular and faster beating frequency(129±11 bpm vs. 82±8 bpm, n=5), a lower Maximal diastolic potential (MDP)(-66±6mV vs.-87±4mV,P<0.05 )than in which myocytes coclutured with HCN4-MSCs. Threshold potential for action potential in all myocytes was -60mV. Immunostaining manifested that CX43 was detected in adjacent membrane between heterologous cell pairs. Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the model lowered spontaneous activity of myocytes in biological pacemaker model.4) Agarose gel electrophoresis for PCR products and gene sequencing confirmed CX45 gene (Gja7) was successfully constructed with pDsRED2-N1-RFP which containing RFP gene. The transfection efficiency for CX45 by the virtue of liposomes 2000 was 60% after 48h. Concentration of G418 for screening CX45+MSCs was 50μg/ml; RT-PCR confirmed the transcription of CX45 gene in CX45+MSCs. Double Immunostaining with CX43 and CX45 antibodies illuminated that CX45 and CX43 colocalized in membrane of CX45+MSCs.5) When connexins in membrane of MSCs was transformed with CX45, a biological pacemaker based on HCN4+CX45+MSCs was characterized with elevated automaticity. Outbreak frequency for spontaneously action potential in biological pacemakers based on HCN4+CX45+MSCs was faster than in control group(147±9 bpm vs. 123±8 bpm, P<0.05).The accelerated VDD in phase 4 mainly contributed to the elevated beating frequency. Double immunostaining manifested that CX43 and CX45 were simultaneously immune-detected in adjacent membrane between heterologous cell pairs. Administration of carbenoxolone to the model lowered spontaneous activity of myocytes in both group to a level identical with native neonatal rat ventricular myocytes.Conclusions: Our findings demonstrate that myocytes cocultured with MSCs modified with HCN4 can act as a cardiac biological pacemaker model in vitro. Overexpression of CX45 will elevate automaticity of cardiac biological pacemakers based on HCN4 modified MSCs in vitro.更多还原