DEVELOPMENT OF GENE THERAPY FOR CARDIOVASCULAR DISEASES

研究了用于心血管系统基因转移的腺病毒载体。国内研究所的作用是建立心血管疾病的体内和体外模型。体外系统包括成年大鼠或新生大鼠心肌细胞、人脐静脉内皮细胞和血管平滑肌细胞。在原代培养中成功制备了基因转移靶细胞。体内系统包括冠脉结扎心肌梗死模型、小鼠心脏移植模型、病毒性心肌炎和心肌病模型，并已建立。长QT综合征模型的进一步建立正在进行中。候选基因有细胞因子、生长因子、离子通道、转录因子等。为了测试这些因子的功能，在各种心肌疾病模型中进行了分子和生化分析，包括northern blotting， western blotting， gel shift assay等。国外合作者构建了腺病毒载体，并对其进行了实验。以含有报告基因-半乳糖苷酶的腺病毒为对照。靶细胞为293细胞、COS细胞或CHO细胞。对照病毒工作良好，重组腺病毒含有感兴趣的cdna，在这些细胞和小鼠中进行了测试。组织型纤溶酶原激活物、组织型纤溶酶原激活物抑制剂、内皮型一氧化氮合酶、内皮前原素作为体内基因转移的候选基因进行了测试。还在培养细胞中测试了溶酶结合腺病毒转移报告基因的能力。在上面列出的所有细胞类型中，都倾向于通过修饰腺病毒成功转移。考虑到该系统对cDNA大小几乎没有限制，而重组腺病毒将其插入片段的大小限制在7kb以内，因此无法整合到重组病毒中的较大基因可能有机会进行基因转移。此外，在与聚酶结合的过程中，腺病毒被灭活，生物危害的风险在该系统中被最小化。使用传统的非病毒载体进行生长因子、细胞因子和离子通道的基因转移，包括它们的原生形式和突变形式。总之，这次合作研究是富有成果和成功的。我们成功地进行了心血管系统修饰和非修饰腺病毒基因转移，用于建立疾病模型、鉴定候选基因、构建载体和检测基因转移。更多基因在体内系统的进一步应用有待继续。少

Adenoviral vectors for gene transfer to cardiovascular systems were investigated.The role of domestic institute was to establish the in vitro and in vivo model of cardiovascular diseases. In vitro system includes cardiac myocytes from adult rats or new born rats, human umbilical venous endothelial cells, and vascular smooth muscle cells. All were successfully prepared in primary culture for gene transfer target cells. In vivo system includes myocardial infarction model by coronary ligation, mouse heart transplant model, viras induced myocarditis and cardiomyopathy model, and have established. Further establishment of long QT syndrome model are on going.For the candidate genes, cytokines, growth factors, ion channels, transcription factors are prepared. To test the functions of these factors, molecular and biochemical analysis including northern blottings, western blottings, gel shift assays, and others are performed in various myocaridal disease models. Adenovirus vectors including mod … More ified form with polylysin or other chemicals were constructed and tested in the oversea collaborators. Adenovirus containing reporter genes, beta-galactosidase were used as a reference. Target cells were 293 cells, COS cells or CHO cells. Control viruses works fine and recombinant adenovirus containing cDNAs of interest were tested in these cells and in mice. Tissue plasminogen activator, tissue plasminogen activator inhibitor, endothelial nitric oxide synthase, preproendothelin are tested as candidate genes for in vivo gene transfers. Polylysin conjugated adenovirus were also tested in cultured cells to transfer reporter genes. In all cell types listed above are prone to successful transfer by modified adenovirus. Considering that this system has vertually no limit in cDNA size while recombinant adenovirus limits its insert size less than 7kb, larger genes which could not be integrated in recombinant virus may have opportunity of gene transfer. Also, adenovirus is inactivated with during conjugation with polylysin, the risk of biaohazard is minimized in this system. Gene transfer with conventional non viral vectors are on going for growth factors, cytokines, and ion channels including thier native forms and mutant forms.In conclusion, this collaborative research was fruitful and successful. Modified and nonmodified adenovirus gene transefer for cardiovascular system was successfully performed for establishing disease models, identifying candidate genes, constructing vectors, and testing gene transfers. Further applications of more genes in in vivo system are to be continued. Less