【作者】 刘丹妮；

【导师】 姚成立；

【作者基本信息】 宁夏医学院 ， 心血管内科学， 2008， 硕士

【摘要】 心脏节律的形成和维持依赖于起搏细胞动作电位4相自动除极化。在动作电位(AP)结束时,心脏起搏细胞去极化使膜电位逐渐增高接近新动作电位启动的阈值,产生周而复始的电活动。作为组织自动除极最主要电流之一,起搏电流(pace maker current,If)无论在自律细胞的起搏[1],还是工作肌细胞异位节律增高方面均起着关键性作用。编码If通道的基因家族在鼠和人类已经被克隆[2,3],分别是HCN1～HCN4,超极化激活环核苷酸门控阳离子通道(HCN)基因是心脏正常起搏活动至关重要的分子基础,起博电流表现为广泛的区域电压依赖性,心脏主要表达HCN2和HCN4[4],在窦房结HCN含量最高的是HCN4占绝对优势的亚型,占总HCN mRNA的80%以上[5,6,7]。本研究旨在创新性的建立系统、简单、适用异源性表达系统,利用全细胞膜片钳技术研究克隆HCN4亚型电生理的特征,明确它对起搏电流(If)的贡献,探讨起搏电流与老年阵发性房颤(PAF)异位起源点的内在联系,揭示If在老年PAF启动中的作用。本课题的目的在于寻找老年PAF起源点产生的机制,为从源头阻断老年PAF奠定理论基础,并为以后药物干预提供新靶点,对于提高老年PAF治愈率,降低病死率将有重要的实用价值。本研究包括以下三个章节,分述如下:第一章起搏通道HCN4质粒的构建及鉴定目的构建HCN4基因和携带绿色荧光蛋白基因(GFP)标记的质粒表达载体pTracer-HCN4-GFP。方法回收质粒DNA片段与载体连接,重组质粒经转化、筛选、提取、鉴定后进行序列测定。同时质粒经双酶切鉴定为目的基因片段。结果成功构建pTracer-HCN4真核表达载体。结论通过酶切鉴定和测序得到证实,起搏通道HCN4基因转入质粒pTracer-CMV2。第二章起搏通道HCN4质粒的转染HEK293细胞进行蛋白mRNA表达目的观察起搏通道HCN4质粒经脂质体瞬时转染细胞进行蛋白表达。方法HCN4质粒在传代培养的HEK293细胞中瞬时转染,通过逆转录聚合酶链反应(RT-PCR)检测转染细胞HCN4基因表达。结果1.在HEK293细胞中脂质体介导mHCN4基因转染成功。2.瞬时转染mHCN4后6～8h,荧光显微镜观察:可见转染细胞的胞核和胞浆区均有GFP表达,发出明亮的绿光;细胞核内表达明显,细胞膜表达为主,核周区也有表达。3.采用RT-PCR方法,扩增出HCN4片段,PCR产物经琼脂糖凝胶电泳分析观察到与预期大小一致的DNA片段。结论脂质体转染后24h,RT-PCR方法和绿色荧光标记基因检测到目的基因及其蛋白的表达,转染后48h较24h蛋白表达有增加的趋势。第三章HCN4质粒在HEK293细胞异源性表达后的电流记录目的观察应用含目的基因HCN4和携带绿色荧光蛋白基因(GFP)标记的质粒表达载体pTracer-HCN4-GFP进行共转染,结合全细胞膜片钳技术记录转染细胞后mHCN4通道电流的特征。方法瞬时转染HEK293细胞行全细胞膜片钳记录。结果记录到一系列超极化内向离子流,该电流呈电压、时间依赖性,没有明显的失活,在-150mV时电流幅值为(540±24.1)pA,半最大激活电位(V1/2)为(-105.7±12.0)mV,稳态激活曲线斜率(k)为-15.7mV,HCN4电流翻转电位为(-84.0±7.9)mV。结论(1)成功构建起搏通道HCN4基因的细胞模型。(2)转染mHCN4基因的HEK293细胞在全细胞膜片钳记录到超极化阳离子电流,加入2mmol/LCsCl后电流几乎完全被阻断提示HCN4质粒转染HEK293细胞不仅有蛋白表达,而且具有起搏通道的功能。更多还原

【Abstract】 Spontaneous electrical activity of the mammalian heart and the adaptation of heart rate by the autonomic tone are physiological key features. Pacemaking activity depends on a phase of spontaneous slow membrane depolarization that occurs before cardiac action potentials during diastolic depolarization phase.In the mammalian cardiac sinus node, the pacemaker current, termed If, plays a crucial physiological role in setting the heart rate and sensing its autonomic control. Hyperpolarization activated cyclic nucleotide gated (HCN) family of ion channels plays a large role in the excitability of heart and nerve cells.The four members of this family, HCN1-4, are similar in structure to voltage-gated potassium channels. Two of the four known genes encoding HCN channel subunits, HCN2 and HCN4,are predominantly expressed in the heart. HCN4 subunits are the molecular components of If channels. Their in vitro properties closely resemble those of native If channels. Therefore, HCN4 gene have been suggested as candidates underlying disorders of heartbeat generation.We determined the functional properties of recombinant HCN4 channels expressed in HEK293 cells. Heterologously expresstion studies of individual HCN4 isoforms have reported in voltage dependence of the expressed isoforms in functional characteristics of If. A transformed line of human embryonic kidney epithelial cells(HEK293) is commonly used as an expression system for exogenous ion channel genes. Patch-clamp electrophysiology experiments confirm the presence of ion channels and molecular data agree with pharmacological profiles of identified channels. This study will offer one of the electrophysiologic mechanisms to treat the aged paroxysmal atrial fibrillation. This study include three parts.Chapter one .Construct the recombinant plasmid HCN4 and identifiedObjective Construct the mouse HCN4 gene and the GFP gene plasmid pTracer-CMV-GFP to form the transfer vector. Methods Recover the plasmid DNA segment and consistent them with pTracer-CMV vector,then put the recombined plasmid into translated,selected,identied for determining the sequencing. Results .We comfirmed that the HCN4 gene enter pTracer-CMV vector for using two enzyme identification and sequencing.Chapter two . Heterologously expresstion transfected the mHCN4 plasmid into HEK293 cellsObjective To transfected the mouse HCN4 gene plasmid into HEK293 line cells and protein expression after transfection by RT-PCR. Methods The mRNA expression was detected by reverse transcriptase-PCR method after being transfected into the HEK293 cell using Lipofactamine2000 reagent for amplification. Results 1 .successfully transfected into the HEK293 cell using Lipofactamine2000 reagent. 2 .There have a lot of bright green light eruption in the cellular nuclear , cytoplasmic domain and cellular member through transient transfection for 6-8 hour. 3 .The expression GFP was observed under fluorescene microscope,and total RNA was abstracted from the HEK293 cells ,the mRNA of HCN4 gene was detected by RT-PCR.Chapter three. Character of HCN4 currents Electrophysiology into HEK293 cellsObjective To transfected the mouse HCN4 gene and the GFP gene plasmid into HEK293 line cells and identify the HCN4 channel Electrophysiology characteristics after gene expression. Methods The HCN4 gene fragment was cloned into the shuttle plasmid pTracer-CMV-GFP to form the transfer vector.The mRNA expression was detected by reverse transcriptase-PCR method after being transfected into the HEK293 cell using Lipofactamine reagent for amplification,then to use whole cell patch clamp recorded. Results An inward current was recorded in response to hyperpolarization activaton in transfected HEK293 cell The mHCN4 channel began to activate in voltage and time dependent manner without deactivation Threshold voltage and potential of half-maximal activation(V1/2) was-70mV,-105.7±12.0mV,respectively.Conclusion 1 .successfully constructed the cell model of pacemaking current that carry HCN4 isoforms. 2. confirmed the expression of HCN4 gene and protein after transfection by RT-PCR. 3. HCN4 gene is heterologously expressed while the less negative activation of HCN4 reported here is consistent with its relative expression in HEK293 cell with less negative activation of native If.The underlying phenomenon may apply comparably to HCN4 since the relative voltage dependence of the isoforms is preserved in the separate expression systems employed in the present study. Elucidation of the factor contributing to the regulation of the voltage dependence of multiple HCN4 isoforms may lead to novel therapies for the aged paroxysmal atrial fibrillation.更多还原