Development of novel mouse models deficient in vasoactive substances-Clinical implication of the natriuretic peptide family and its application to gene therapy-

血管活性物质缺乏的新型小鼠模型的开发-利尿钠肽家族的临床意义及其在基因治疗中的应用-

基本信息

批准号：
07557072
负责人：
NAKAO Kazuwa
金额：
$ 7.17万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

The mouse BNP and CNP genes were isolated from a 129Sv mouse genomic library. Targeting vectors for the disruption of BNP or CNP were constructed, in wihch the 2nd and 3rd exons of the BNP gene or the 1st exon of the CNP gene were replaced by the neomycin resistance gene. Several chimeric mice were obtained from the targeted ES cell lines. We are currently mating heterozygotes to obtain mice that are homozygous for the disrupted allele.We characterized a genomic DNA fragment containing the ANP and BNP genes in mice and humans. In mice, the BNP gene was located about 12kb upstream of the ANP gene. An 11-kb human genomic DNA fragment was isolated, which contained the 3rd exon of the BNP gene and the 1st and 2nd exons of the ANP gene, approximately 8kb apart. Therefore, ANP and BNP genes are organized in tandem in mice and humans.We examined BNP gene expression in cultured neonatal rat ventricular cardiocytes. During ET-1-induced cardiocyte hypertrophy, BNP mRNA was induced more rapidly t … More han ANP mRNA.BNP secretion was also stimulated more rapidly than ANP secretion. Furthermore, BNP mRNA turnover was significantly earlier than ANP mRNA turnover. These results demonstrate that BNP gene expression is distinctly regulated from ANP gene expression at transcriptional and posttranscriptional levels, suggesting the possible role of BNP as an "emergency" cardiac hormone against ventricular overload.We examined the interaction of endothelial cells (ECs) and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth. The data indicate augmented production of CNP with the intracellular cGMP accumulation in the EC/SMC coculture. Biologically active TGF-beta in the coculture with direct contact of ECs and SMCs stimulated endothelial productin of CNP.Furthermore, the culture medium from ECs stimulated by TGF-beta had a growth-inhibitory effect on SMCs. These results indicate that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-beta, suggesting the pathophysiological significance of CNP as a regulator of vascular growth in the interaction of ECs and SMCs. Less

从129SV小鼠基因组文库中分离出小鼠BNP和CNP基因。在WIHCH中构建了破坏BNP或CNP的靶向载体，BNP基因的第二和第三外显子或CNP基因的第一个外显子被Neomycin耐药基因取代。从靶向的ES细胞系获得了几只嵌合小鼠。我们目前正在交配杂合子，以获取对破坏等位基因纯合的小鼠。我们表征了一个基因组DNA片段，该基因组DNA片段中包含小鼠和人类中的ANP和BNP基因。在小鼠中，BNP基因位于ANP基因上游约12KB。分离了11 kb的人基因组DNA片段，其中包含BNP基因的第三外显子，而ANP基因的第一个和第二个外显子分开了约8KB。因此，在小鼠和人类的串联中组织了ANP和BNP基因。我们检查了培养的新生大鼠心室心脏细胞中BNP基因的表达。在ET-1诱导的心脏肥大中，BNP mRNA的诱导更快地诱导了……比ANP分泌更快地刺激了HAN ANP mRNA.BNP。此外，BNP mRNA更新明显早于ANP mRNA更新。这些结果表明，BNP基因表达在转录和转录后水平上明显调节ANP基因表达，这表明BNP作为对心室过载的“紧急”心激素的可能作用。我们检查了内皮细胞（ECS）（ECS）的相互作用（ECS）和血管平滑肌细胞（SMCS）的作用，以实现CNP的作用。数据表明，在EC/SMC共培养中，CNP的产生增加了CNP。共培养中具有ECS和SMC的直接接触的生物活性TGF-β刺激了CNP的内皮产物。Furthermore，TGF-β刺激的ECS的培养基对SMC具有生长抑制作用。这些结果表明，CNP在EC/SMC共培养中的内皮产生至少部分受TGF-beta调节，这表明CNP作为EC和SMCS相互作用中血管生长的调节剂的病理生理意义。较少的