【作者】 王军；

【导师】 王运杰；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 博士

【摘要】 目的电压-门控Na+通道(VGSC)是可兴奋细胞动作电位产生和传导的重要离子通道,它有一个可以单独发挥作用的α亚单位(240-260kD)和1-4个起辅助作用的β亚单位(β1-β4,33-36kD)构成。目前为止,已有10个Na+通道α亚单位(Nav1.1-Nav1.9,Nax)被成功克隆,根据其对河豚毒(TTX)敏感与否,VGSC可分为两类,一类是河豚毒敏感型(TTX-S)Na+通道,包括Nav1.1,Nav1.2,Nav1.3,Nav1.4,Nav1.6和Nav1.7Na+通道,另一类对河豚毒抵抗(TTX-R)或不敏感(TTX-I),包括Nav1.5,Nav1.8和Nav1.9 Na+通道。其中TTX-S型的Nav1.1,Nav1.2和Nav1.3 Na+通道在神经元中分布广泛并首先在鼠脑组织中被成功克隆,故被称为脑型Na+通道,而河豚毒-抵抗(TTX-R)的Nav1.5Na+通道被认为是心肌特异性Na+通道,与心脏电活动密切相关,而且已有许多心脏疾患被证明是Nav1.5／SCN5A基因的突变所致,但最近的研究发现神经元中也存在TTX-R型Na+电流,并且可以检测到参与编码TTX-R型Na+通道的Nav1.5／SCN5A基因的表达,所以提示神经元中也存在TTX-R型Nav1.5Na+通道,但编码该离子通道的确切基因序列尚不清楚,它与心肌Nav1.5Na+通道结构和功能的差异也不明了,所以本实验旨在对人脑Nav1.5／SCN5A基因进行全序克隆,以期发现人脑组织Nav1.5 Na+通道与心肌组织该离子通道的编码基因的异同,同时对Wistar大鼠全身16种组织中Nav1.5 Na+通道编码基因Nav1.5／SCN5A的表达情况进行检测和分析,并研究其随发育周期的变化趋势,进而为离子通道病的诊断及治疗提供理论依据。材料与方法一、材料人脑组织来源于中国医科大学附属一院神经外科颅底脑膜瘤患者行肿瘤切除术中内减压额叶(皮质)。健康雄性Wistar大鼠(P1-P120)由中国医科大学实验动物中心提供。总RNA提取及RT-PCR试剂盒有TaKaRa (Japan)公司提供,凝胶回收及质粒提取试剂盒由QIAGEN公司(USA)提供,PGEM-T载体来自Promega公司(USA),基因测序用3700自动基因测序仪(USA)。本研究符合Helsinki宣言的要求并得到中国医科大学伦理委员会的批准和实验动物中心的认可。二、方法(一)总RNA的提取及RT-PCR采用Trizol试剂一步法提取人脑和鼠脑组织总RNA,紫外分光光度计测定纯度并定量,分别进行反转录合成cDNA,参考人心肌Nav1.5/SCN5A基因序列(hH1(accession nos M77235))设计可以扩增人脑Nav1.5/SCN5A基因全序的10对引物(P1-P10)进行PCR反应,得到10段(相邻片断之间有部分重叠序列)人脑Nav1.5/SCN5A基因片断,每段RT-PCR反应至少重复3次。(二)基因克隆每段RT-PCR产物经凝胶回收纯化后,连到PGEM-T载体,而后转化到感受态细胞中进行克隆扩增,提取质粒后自动基因测序仪测序,去除中间部分重叠序列,得到人脑组织Nav1.5/SCN5A基因cDNA全序。(三)竞争性PCR为研究新的选择性剪切体的表达比率,应用竞争性PCR,即改变PCR的循环次数,而其它反应条件保持不变。(四)统计学分析反应结束后,凝胶图像分析系统进行灰度分析,每种选择性剪切体的表达值以(变构体的吸光度值/β-actin吸光度值)*100%表示,结果分析以均数±标准差表示。大鼠不同组织间Nav1.5/SCN5A基因表达的差异表达用t检验(Student’s t-tests),P<0.05认为有统计学意义。结果一、参与编码人和鼠脑组织Nav1.5Na+通道的是Nav1.5/SCN5A基因的第6A外显子而非第6外显子特异性的扩增第6(eXon6)及第6A(exon6A)外显子的引物进行PCR反应,结果显示人脑和鼠脑组织中有exon6A的表达而无exon6的表达。RT-PCR产物纯化后进行克隆测序,再次证明是exon6A而非exon6参与编码脑组织Nav1.5 Na+通道。exon6和exon6A都有92个碱基,都可以编码产生30个氨基酸,这些氨基酸都位于Nav1.5 Na+通道α亚单位的D1-S3-S4区域,但两者编码产生的这些氨基酸却有7个不相同。二、人脑组织Nav1.5／SCN5A基因的全序克隆及与hH1和hNbR1的比较人脑组织Nav1.5/SCN5A基因cDNA有两种变构体,分别命名为hB1和hB2,其中hB1全长6201个碱基,其开放读码框架(ORF)参与编码2016个氨基酸,和人心肌Nav1.5 Na+通道氨基酸序列(hHl)相似率高达98%,共有28个不同氨基酸,其中的7个集中位于第6A和第6外显子编码区,其余不同氨基酸分布情况如下：DⅠ区16个,LoopⅠ-Ⅱ区2个,DⅡ区1个,LoopⅡ-Ⅲ区5个,DⅢ区1个,LoopⅢ-Ⅳ区0个,DⅣ区3个。和人神经母细胞瘤细胞Nav1.5 Na+通道氨基酸序列(hNbRl)相比,二者相似率更高,仅有20个不同,分布情况如下：DⅠ区8个,LoopⅠ-Ⅱ区2个,DⅡ区1个,LoopⅡ-Ⅲ区4个,DⅢ区1个,LoopⅢ-Ⅳ区0个,DⅣ区4个。三、Nav 1.5/SCN5A基因的mRNA在不同发育阶段大鼠中枢神经系统不同部位中的表达及变化趋势Nav1.5 Na+通道mRNA在中枢神经系统各部位表达高低不同,在不同周龄大鼠大脑组织中的相对表达值分别为：(P1(82.11±7.29)%；P9(72.37±6.14)%；P40(66.73±5.17)%；P80(52.42±5.01)%；P120(48.34±3.73)%)。在小脑组织中的相对表达值:(P1(76.62±6.83)%；P9(68.21±6.13)%；P40(55.42±4.64)%;P80(46.03±4.08)%；P120(42.24±3.86)%)。在海马组织中的相对表达值分别为：(P1(80.12±7.34)%；P9(74.22±6.02)%；P40(65.85±5.29)%；P80(58.43±5.21)%；P120(50.21±4.85)%).在脑干组织中的相对表达值分别为：(P1(75.28±6.41)%；P9(70.16±6.03)%；P40(60.44±5.82)%；P80(50.11±4.76)%；P120(45.03±3.75)%)。在成鼠神经系统各部位的表达高低为海马>大脑>脑干>小脑>脊髓,但随着周龄的增加,大脑、小脑、海马、脑干和脊髓中该通道的表达值均呈下降趋势。四、Nav1.5 Na+通道在Wistar大鼠全身各组织中的分布利用RT-PCR法Wistar成鼠(P80)全身16种组织中Nav1.5/SCN5A基因mRNA进行检测发现Wistar大鼠全身16种组织中均可检测到Nav1.5/SCN5A基因mRNA的表达,但不同组织中Nav1.5/SCN5A基因mRNA的表达高低不同,其中以心肌组织中的表达为最高,脑组织、脊髓和睾丸组织次之,肺、脾、骨骼肌、胃、小肠、大肠、肾上腺和膀胱中中度表达,肝和胰腺组织中低表达。结论1、人脑组织Nav1.5 Na+通道与心肌组织该离子通道的编码基因不同,是Nav1.5/SCN5A基因的两种变构体,且该变构体的分布具有神经组织特异性。2、人脑组织Nav1.5/SCN5A基因存在2种新的选择性剪接体,分别为第6A和6外显子(exon6A和exon6)的选择性剪接,以及第24外显子(exon24)的选择性剪接,不同选择性剪接体在不同组织中的表达量不同。3、中枢神经系统不同部位Nav1.5 Na+通道的分布不同,以幼鼠的海马及大脑皮层为多,但随着年龄的增长,不同部位的表达均呈下降趋势。4、Nav1.5 Na+通道的分布十分广泛,全身各系统中几乎均可见Nav1.5 Na+通道的分布,但其表达量不同,其编码基因是否完全相同仍不清楚,所以其在不同组织中的具体功能及与离子通道病的关系值得深入研究。更多还原

【Abstract】 ObjectiveVoltage-gated sodium channels (VGSC), which play an essential role in the generation and propagation of action potentials, consist of a large pore-forming a-subunit (≈260kDa, Navl) associated with smaller auxiliaryβ-subunits,β1 (36kDa),β2 (33kDa),β3 andβ4. According to their binding affinity to the specific inhibitor TTX, voltage-gated sodium channels are classified into two types. One type is sensitive to TTX, including Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6 and Nav1.7, and the other type is resistant or insensitive to TTX, including Navl.5, Nav1.8 and Nav1.9. Among those channels, Nav1.1, Nav1.2 and Navl.3 were first cloned from the brain and functionally analyzed, as they were originally considered as brain sodium channel typesⅠ,ⅡandⅢ. As mentioned above, these channels are sensitive to TTX, interestingly, Na+currents with TTX resistance have also been observed in neurons, but the full-length genes corresponding to these currents in central nervous system (CNS) remain unknown. Although PCR amplified moderate levels of the Navl.5 mRNA from human cerebral cortex, the full-length Nav1.5 cDNA of the brain has not been identified and its specific distribution among different part of the brain was unknown either, let alone its expression patterns with age development. Navl.5 channel was first cloned from the heart and has been well studied, but the structural and functional differences between cardiac Navl.5 channels and cerebral Navl.5 channels still remain unknown, so one of the aims of this study was to clone the full-length Navl.5 cDNA from the brain in order to find the differences between them. Interestingly, our results show that Nav1.5 channels in human and rat brains are encoded by new variants of Nav1.5/SCN5A gene and this gene is more widely distributed and expressed than previously thought.Materials and methods1. MaterialsThe investigation was approved by the Ethic Committee and the Committee of Animal Experimentation of China Medical University and conformed to the principles outlined in the declaration of Helsinki. Human brain tissue was obtained from the human frontal lobe which was dissected for curing basilar meningioma at the first hospital affiliated to China Medical University. And the experiment was undertaken with the understanding and written consent of the patient. Healthy male Wistar rats at different developmental stages (P1-P120) were provided by the Animal Experimentation Center of China Medical University. The rats were anaesthetized by inhalation of a mixture of 50% O2 and 50% CO2 and killed by cerebral dislocation, with all tissues used carefully excised.2. Methods(1) RNA isolation and RT-PCRTotal RNA was extracted from all tissues using RNA out kit (TaKaRa, Japan) according to the manufacture’s instructions. The first-strand cDNA was synthesized using the RT-PCR kit (TaKaRa, Japan) with oligo-(dt) and random primers. The PCR primers for the amplification of Navl.5 and Navβ1 were designed from highly conserved sequences among species but not among isoforms and the PCR was carried out according to its instructions. All PCRs for detecting the relative amount of Nav1.5 were repeated at least three times.(2) Cloning of Nav1.5 cDNAFor the isolation of a variant of Nav1.5 from human brain, we used 10 primer pairs to amplify ten fragments of the full length cDNA by the PCR method described above. All of the ten fragments partially over-lapped each other allowing subsequent assembly using common restriction enzyme sites. The PCR products (300-1300bp) were separated on 1-2% agarose gel, and fragments of the expected size were extracted by using gel extraction kit (Qiagen, USA) and then were subcloned into the PGEM-T.easy vector (Promega, USA) and sequenced by using 3700 DNA sequencer(USA) after plasmid extraction(Qiagen, USA). Multiple independent recombinants of each construct were sequenced completely to identify clones without polymerase errors.(3) Statistical analysisPCR products were analyzed by gel electrophoreses (1%-2% agarose).The signal of each band was determined using Quantity One 4.5 software (USA). For normalization of the amount of cDNA, we usedβ-actin and GAPDH as an internal standard. So the gene expression levels were presented as amplicon densities toβ-actin or GAPDH and data for gene expression values were presented as means±S.E.M. Student’s t-tests were used as appropriate to evaluate the statistical significance of differences between two group means, and analysis of variance used for multiple groups. Values of P＜0.05 were considered to indicate statistical significance.Results1. It is exon6A rather than exon6 of Nav1.5/SCN5A that encodes Nav1.5 channels in the brainIn order to investigate whether exon6 and/or exon6A encode(s) Nav1.5 channels in the brain, special primer pairs for amplification of exon6A and exon6 were designed. The PCR products were separated by electrophoresis onl.5% agarose gel after 35 cycles. The expected fragment of exon6 was not found from the brain cDNAs and that of exon6A was not found in non-brain tissues, such as heart, lung and testis. Sequence analysis further confirmed that it was exon6A rather than exon6 that encoded Navl.5 channels in the brain. Both exon6A and exon6 had 92 base pairs, which encoded 30 amino acid residues. But 7 amino acid residues were ifferent between them. There was only one different nucleotide between the exon6A of human Navl.5/SCN5A and that of the rat or human neuroblastoma cell Nav1.5/SCN5A, but they encode identical amino acid residues.2. Cloning of Nav1.5 channel a-subunit from the brainTo obtain the full-length cDNA encoding Navl.5 in the human cerebral cortex, ten primer pairs (table 1) were designed based on the published human hHl (accession no: M77235) sequence, which predicted fragment sizes ranging between 300bp to 1300bp. Then ten different sub-regions of the full-length cDNA were separately amplified using PCR method.In the study, four full-length cDNAs encoding the a-subunits of the Navl.5 channels in human and rat cerebral cortexes were found and cloned, which were designated hB1, hB2, rN1 and rN2. The full size of the human Navl.5 cDNA was 6201 nucleotide long and was designated hBl.The longest open reading frame of hBl encoded 2016 a.a residues and it was highly homologous with hHl (>98% a.a identity) and hNbRl (>99% a.a identity).The hBl differed from hHl by 28 a.a. They were distributed in domainⅠ(16), loop 1-Ⅱ(2), domainⅡ(1), loopⅡ-Ⅲ(5), domainⅢ(1), loopⅢ-Ⅳ(0), domainⅣ(3). However, when compared with hNbRl, there was 1996 a.a. identity with only 20 a.a difference. They were distributed in domain I (8), loopⅠ-Ⅱ(2), domainⅡ(1), loopⅡ-Ⅲ(4), domainⅢ(1), loopⅢ-Ⅳ(0), domain IV (4).The hB2 was 6147 nucleotides long, lacking exon24 compared with hB1, and encoded 1998 amino acid residues. The missing exon24 encodes 18 amino acids in the extracelluar loop between S5 and S6 of domain III.The open reading frame of rNl also encoded 2016 amino acid residues and sequence analysis indicated that it was highly homologous with mHlwith>96% amino acids identity. Alternative splicing of exon24 was also found in the cloning of Nav1.5/SCN5A from the rat brain and the new variant was designated rN2, which encoded 1998 amino acid residues. There were four different nucleotides between exon24 of human Nav1.5/SCN5A and that of rat Nav1.5/SCN5A, but they encoded identical amino acid residues. 3. The expression patterns of Nav1.5 mRNA in different part of developing rat brainsIn order to investigate whether the expression levels of Nav1.5 mRNA in CNS change with age development, rat brains and spinal cords at five different developmental stages (PI, P9, P40, P80, P120) were used. We detected the expressions of Nav1.5 mRNA in cerebral cortex, hippocampus, cerebellum, brain stem and spinal cord (cervical) using RT-PCR method. The results indicated that the expression levels of Nav1.5 mRNA in different part of the brain showed a similar expression pattern with age development. In cerebral cortex, hippocampus, brain stem and cerebellum, the high expression levels were detected in neonatal rats (P1, P9), whereas expression decreased with age development up to P120.4. The expression of total Nav1.5 mRNA in 16 different tissue types of Wistar ratsRT-PCR method was used to detect the expression of total Nav1.5 mRNA in 16 different tissue types of Wistar rats (P80). As reported previously, Navl.5 mRNA showed the strongest expression in the heart. And our results indicated that it could also be detected in all tissues assayed. Compared to its extremely high expression in the heart, the Nav1.5 mRNA showed high expression levels in the adult and fetal rat brains, spinal cord and testis, moderate levels in the kidney, adrenal gland, lung, skeletal muscle, spleen, stomach and bladder, and low levels in the liver and pancreas.Conclusion1. Nav1.5 channels in the brain are encoded by new variants of Nav1.5/SCN5A> but their functions remain unknown.2. Two novel alternative splicing variants of Nav1.5/SCN5A are found in our study (alternative splicing of exon6A/exon6 and exon24). And their expression levels in different tissues are different.3. The expression levels of Nav1.5 mRNA in different part of the brain are different, and the high expression levels were detected in neonatal rats (PI, P9), whereas expression decreased with age development (up to P120).4. Nav1.5 channels are more widely distributed and expressed than previously thought, but its specific encoding genes and functions in different tissues remain unknown, and the relationship between Navl.5 channels and ion channel diseases merits further investigation.更多还原