Functional analysis of nitric oxide synthase related genes transfered into cardiomyocytes in viv

方法将内皮型一氧化氮合酶(内皮型一氧化氮合酶，ecNOS)基因导入活体大鼠心肌细胞内，检测一氧化氮在活体心脏的直接作用。结果1)标记基因β-Gal(β-半乳糖苷酶)基因在3天后表达，达到最高水平，14天后仍有表达。在无HVJ-loposome的情况下，基因转移效率低于10%。用HVJ-loposome方法进行的β-Gal基因转移没有引起炎症反应，并且基因定位在插入盘内的范围使我们能够区分通过基因转移而发生的细胞变化和同一细胞的未转染部分。ecNOS基因转移到活体Geart中，意外地引起了严重的组织损伤。心肌组织损伤与β-半乳糖和ecNOS转染区共定位。基因转移量较少(5微克对50微克)引起的组织损伤较轻。预先给予一氧化氮合酶抑制剂L-NAME可抑制内皮型一氧化氮合酶所致的组织损伤。组织损伤区有巨噬细胞聚集和纤维化，有少量TUNEL阳性细胞。电子显微镜下可见肌瘤周围线粒体聚集增大、变形。讨论将一氧化氮合酶基因转移到活体心脏，有望保护心脏免受组织损伤所致的严重心脏损伤。分析了引入的一氧化氮合酶用量和周围环境条件的影响。

MethodsEndothelial nitric oxide synthase(ecNOS)gene has been tranafered into in vivo rat cardiomyocytes with combination of HVJ(Hemagglutinating virus of Japan)and liposomes to examine the direct of nitric oxide in a living heart.Results1)The expression of the marker gene, beta-gal(beta-galactosidase)gene was detected 3 days after the gene transfer, reached the maximal level, and still remained 14 days after the in vivo transfer. The efficiency of the gene transfer without HVJ-loposome was less than 10%. The beta-gal gene transfer with HVJ-loposome method did not induce inflammatory findings, and the extent of the gene localized within a intercalated disc which enabled us to distinguish the cellular change by gene transfer from untransfected parts of the same cell.ecNOS gene transfer into a living geart induced severe tissue damage, unexpectedly. The cardiac tissue damage co-localized with beta-gal and ecNOS transfected area. The gene transfer of less amount(5mug vs.50mug)evoked milder tissue injury. Pretreatment of ^<L->NAME,a NOS inhibitor, suppressed the tissue damage by ecNOS transfection. The area of tissue damage contained accumulation of macrophages and fibrosis with a few area of TUNEL-positive cells. Electronmicroscope showed enlarged and deformed mitochondrial accumulation beside disarray of sarcomeres.DiscussionsecNOS gene transfer into a living heart which was expected to protect heart from tissue injury induced severe cardiac damage. Influence of the introduced NOS amount or of surrounding condition has been analyzed.