DEVELOPMENT OF GENE THERAPY FOR CARDIOVASCULAR DISEASES

针对心血管系统基因转移的腺病毒载体进行了研究，国内研究机构的作用是建立体外和体内心血管疾病模型。体外培养体系包括成年大鼠和新生大鼠的心肌细胞、人脐静脉内皮细胞和血管平滑肌细胞。均在原代培养中成功制备，可作为基因转移的靶细胞。体内系统包括冠脉结扎法心肌梗死模型、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，针对候选基因，制备了细胞因子、生长因子、离子通道、转录因子等。为了验证这些因子的功能，在不同的心肌病模型中进行了包括Northern杂交、Western blotting、凝胶位移分析等在内的分子和生化分析。包括Mod…在内的腺病毒载体用多聚赖氨酸或其他化学物质构建了更具体化的形式，并在海外合作者中进行了测试。以含报告基因的腺病毒、β-半乳糖苷酶为参照。靶细胞为293细胞、COS细胞或CHO细胞。对照病毒工作良好，含有感兴趣的cDNA的重组腺病毒在这些细胞和小鼠身上进行了测试。组织型纤溶酶原激活剂、组织型纤溶酶原激活剂抑制物、内皮型一氧化氮合酶、前内皮素作为体内基因转移的候选基因。多聚赖氨酸偶联腺病毒也在培养细胞中进行检测，以转移报告基因。在以上列出的所有细胞类型中，都容易通过改良腺病毒成功转移。考虑到该系统对cDNA大小没有限制，而重组腺病毒将其插入片段限制在7kb以内，不能整合到重组病毒中的较大基因可能有基因转移的机会。此外，在与多聚赖氨酸结合的过程中，腺病毒被灭活，在这个系统中，生物危害的风险被降到最低。传统的非病毒载体的基因转移正在向生长因子、细胞因子和离子通道转移，包括它们的天然形式和突变形式。修饰和未修饰的心血管系统腺病毒基因转移成功地用于疾病模型的建立、候选基因的识别、载体的构建和基因转移的检测。更多的基因在体内系统中的进一步应用还有待继续。较少

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