【作者】 孙莉萍；

【导师】 浦介麟； 方丕华； 腾思勇；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 核纤层蛋白基因突变可以引起扩张型心肌病,其特点是以房室传导阻滞为首发症状,心脏扩张症状出现迟于电生理异常；患者猝死率高,死因常为恶性心律失常。其发病机制现在尚不清楚,本研究从分子电生理机制方面探讨传导阻滞首发的原因,为疾病表型的发生机理提供一个可能的假说。第一部分遗传性扩张型心肌病LMNA基因突变体表达载体的构建目的：构建遗传性扩张型心肌病LMNA基因E82K、R644C、N195K突变体表达载体。方法：利用快速PCR定点突变技术,设计一对引物(包含预定的突变),通过PCR扩增出含突变位点的质粒,然后转化到超级感受态细胞中,用碱裂解法抽提质粒,进行测序鉴定,得到构建遗传性扩张型心肌病LMNA E82K、R644C、N195K突变体HEK293细胞异源表达载体；通过测序验证突变的正确性。结果：测序验证我们成功构建了遗传性扩张型心肌病LMNA基因E82K、R644C、N195K突变体表达载体。结论：成功构建表达载体为下一步研究奠定基础。第二部分致遗传性扩张型心肌病房室伴传导阻滞的核纤层蛋白基因突变下调钠电流密度目的：传导阻滞和恶性心律失常常与钠通道障碍有关。因此,本部分研究核纤层蛋白基因突变对钠通道功能的影响。方法：采用聚合酶链反应法制备相关LMNA突变体：E82K、R644C和N195K。将突变和野生的LMNA突变体和SCN5A的cDNA共转到HEK293细胞,应用全细胞膜片钳技术记录通道电流；利用实时定量RT-PCR和Western blot分析SCN5A基因表达水平。结果：相比于野生型,LMNA E82K明显降低HEK293细胞钠电流密度LMNA(86.19±9.36pA／pF,n=30 vs.155.73±19.09pA／pF,n=47;p=0.001),而LMNA R644C和N195K对此无明显影响。LMNA突变体不影响SCN5A的转录水平,但是LMNA E82K会抑制SCN5A蛋白表达,幅度达到30%。其他两个突变对蛋白表达水平并无影响。结论：核纤层蛋白导致的钠电流密度减小可能是核纤层蛋白突变致心脏传导阻滞的机制之一,它通过抑制蛋白表达水平抑制电流,而不影响转录水平;但它并非是唯一机制,LMNA N195K可能通过其它机制引起临床上传导阻滞的疾病表型。第三部分致遗传性扩张型心肌病伴房室传导阻滞的核纤层蛋白基因突变引起缝隙连接43重构目的：核纤层蛋白基因突变引起的扩张型心肌病的特点是以房室传导阻滞为首发症状,心脏扩张症状出现较迟。这种疾病表型与缝隙链接蛋白异常引起的心脏疾病相似。本部分研究核纤层蛋白基因突变对缝隙链接蛋白的影响。方法：利用野生和突变(E82K和R644C)的核纤层蛋白基因转染乳鼠心肌细胞,通过共聚焦显微镜和蛋白印迹分析探索缝隙链接改变。结果：E82K突变使缝隙链接蛋白43的表达量减少40%,其定位也发生改变,由细胞连接处转移到细胞内；但该突变对缝隙链接蛋白40影响不显著。而R644C突变对缝隙链接蛋白43和40都无明显影响。结论：核纤层蛋白基因突变导致的缝隙链接蛋白表达和定位的改变可能是导致心脏传导阻滞表现的机制之一。更多还原

【Abstract】 Mutations in the lamin A/C gene (LMNA) may cause familial dilated cardiomyopathy (DCM) characterized by early onset of atrioventricular block (AVB) before the manifestation of DCM. The molecular mechanisms underlying the disease have not been fully clarified. We suggest a hypothesis that it is due to abnormalities of SCN5A ion channel and gap junctions explored using molecular biology and patch clamping techniques.Part 1 Construction of the Eukaryotic Expression Vector of the LMNA Gene Mutation E82K、R644C、N195KObjective:To construct the eukaryotic expression vector of the HERG gene and express in HEK293 cell. Methods:Amino acid substitution of glutamic for aspartic at position 82、644 and 195(E82K、R644C、N195K)in LMNA was performed by polymerase chain reaction(PCR).Mutagenesis was performed by site-directed mutagenesis PCR. The primers contained the mutant site. The mutant plasmid was confirmed by sequence analysis. Results:The eukaryotic expression vector was constructed correctly. Conclusions:The constrauts was made successfully, it was the foundation for the further functional study. Part 2 Familial dilated cardiomyopathy-related lamin A/C gene mutation E82K reduces sodium current densityObjective:The phenotype of LMNA-related DCM is very similar to the phenotype of SCN5A-related DCM. Therefore, we studied the effects of LMNA mutations on SCN5A channels. Methods and results:The sodium current densities were studied in HEK293 cell lines co-transfected with wild-type (WT) or mutant LMNA (E82K and R644C) and SCN5A cDNA using whole-cell patch-clamp recording techniques. The peak sodium current density was reduced significantly in the cells transfected with LMNA E82K compared to that in cells transfected with WT LMNA (86.19±9.36pA/pF, n=30 vs.155.73±19.09pA/pF, n=47; p=0.001). The reduction of sodium current densities in LMNA E82K mutant cells was due to repressed SCN5A gene translation. The level of sodium channel protein was 0.72±0.14 in LMNA E82K cells relative to control vs.1±0.13 in WT LMNA cells (p<0.05); however, transcription of the SCN5A gene was not altered. Conclusions:Our findings suggest that the LMNA E82K mutation down-regulates sodium channel function by inhibiting SCN5A translation rather than transcription, and provides new insights into the mechanisms of DCM in these patients. The exact mechanism of the effect of the LMNA E82K mutation on SCN5A translation remains to be elucidated.Part 3 Connexin43 Remodeling induced by LMNA gene mutations in family Dilated Cardiomyopathy with atrial ventricular blockObject:Mutations in the lamin A/C gene (LMNA) may cause familial dilated cardiomyopathy (dilated cardiomyopathy) characterized by early onset atrio-ventricular block (A-V block) before the manifestation of dilated cardiomyopathy and high risk of sudden death due to ventricular arrhythmia, which is very similar to the phenotype of gap junction related heart disease. In this part, we studied the effect of LMNA mutations on connexins. Methods:The Cx43 and Cx40 expression in cultured neonatal myocytes transfected with wild-type (WT) or mutant LMNA (E82K and R644C) were studied using confocal imaging and western blot analysis. Results:Cx43 protein expression was reduced by 40% in cells transfected with LMNA E82K than that in cells transfected with WT LMNA cDNA. Confocal imaging showed that the Cx43 located inside of the cells. By contrast, LMNA E82K mutation had no effect on expression and localization of Cx40. LMNA R644C transfection did not show any significant effects on gap junctions at all. Conclusions--Our findings suggest that LMNA E82K significantly reduced the Cx43 expression and altered its localization which may be one of the pathological mechanisms underlie LMNA-related heart disease.更多还原