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

从129Sv小鼠基因组文库中分离得到小鼠BNP和CNP基因。将BNP基因的第2、3外显子或CNP基因的第1外显子替换为新霉素抗性基因，构建了BNP或CNP基因打乱的靶向载体。从靶向ES细胞系中获得了几只嵌合小鼠。我们目前正在对杂合子进行配对，以获得对被破坏的等位基因纯合的小鼠。我们在小鼠和人类中鉴定了包含ANP和BNP基因的基因组DNA片段。在小鼠中，BNP基因位于ANP基因上游约12kb。克隆了一段11kb的人类基因组DNA片段，该片段含有BNP基因的第3外显子和ANP基因的第1、2外显子，相距约8kb。因此，在小鼠和人类中，ANP和BNP基因被串联在一起。我们检测了BNP基因在培养的新生大鼠心肌细胞中的表达。在ET-1诱导的心肌肥大过程中，BNP…的表达速度更快。更多的汉族人ANP mRNA.BNP的分泌也比ANP的分泌更快。此外，BNP信使核糖核酸周转明显早于心钠素信使核糖核酸周转。这些结果表明，BNP基因的表达在转录和转录后水平上明显受ANP基因表达的调控，提示BNP可能是一种对抗心脏超负荷的“紧急”心脏激素。我们研究了内皮细胞(ECs)和血管平滑肌细胞(SMCs)对CNP内皮产生的相互作用及其对血管生长的作用。结果表明，在EC/SMC共培养过程中，随着细胞内cGMP的积累，CNP的产量增加。内皮细胞与血管内皮细胞直接接触的共同培养中，具有生物活性的转化生长因子-β刺激内皮细胞产生内皮细胞，且内皮细胞培养上清液对血管内皮细胞有生长抑制作用。这些结果表明，在EC/SMC共培养中，CNP的内皮产生至少部分受转化生长因子-β的调节，提示CNP在ECs和SMC相互作用中作为血管生长调节因子的病理生理意义。较少

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