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

方法采用日本血凝病毒（HVJ）与脂质体联合转染内皮细胞一氧化氮合酶（ecNOS）基因，观察一氧化氮在活体心脏中的直接作用。结果1)标记基因β -半乳糖苷酶（β -半乳糖苷酶）基因的表达在基因转移后第3天检测到，达到最高水平，在体内转移后第14天仍保持不变。无hvj染色体的基因转染效率低于10%。用HVJ-loposome方法转移β -半胱氨酸基因不会引起炎症，并且基因定位在嵌入盘内的程度使我们能够将基因转移引起的细胞变化与同一细胞未转染的部分区分开来。出乎意料的是，将ecNOS基因转移到活体器官会引起严重的组织损伤。心脏组织损伤与β -gal和ecNOS转染区共定位。基因转移量较少（5mug vs.50mug）引起的组织损伤较轻。预处理NOS抑制剂^<L->NAME可抑制ecNOS转染引起的组织损伤。组织损伤区巨噬细胞聚集，纤维化，少量tunel阳性细胞。电镜下可见肌节紊乱旁线粒体堆积肿大、变形。secnos基因转移到活体心脏中，有望保护心脏免受组织损伤引起的严重心脏损伤。分析了氮氧化物引入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.