Creation and analysis of transgenic and knockout animals in hypertension and its related diseases.

本课题的重要研究成果将在以下两部分进行总结。1)血管紧张素原基因敲除小鼠的功能分析缺乏血管紧张素原（Agt-KO）的基因改变小鼠表现出预期的表型，如明显的低血压，但意外的表型包括肾脏形态异常、新生儿存活率降低、冷伤后血脑屏障功能受损。为了研究血管紧张素原基因的破坏是否导致了所观察到的表型，我们在小鼠金属硫蛋白- 1启动子（MT-Agt）的控制下产生了小鼠血管紧张素原基因杂合的小鼠系，并将转基因小鼠与Agt-KO小鼠杂交。由此产生的mic (MT-Agt（+/-)/Agt(-/-): MT-Agt/KO）产生与野生型小鼠相当的血浆血管紧张素I水平，并且突变表型被挽救。2)血管紧张素原敲除小鼠的新功能血管紧张素原敲除小鼠（Ag-KO）基因系形态学分析显示，Ag-KO小鼠海马和小脑颗粒层细胞密度降低，提示Ag-KO神经元细胞易发生凋亡细胞死亡。通过降低血清浓度的海马神经元培养进一步研究了这一现象。Ag-KO神经元细胞在低浓度血清作用下在数小时内出现凋亡，而在血管紧张素II （All）和IV（AIV）存在下存活时间更长。这种抗凋亡作用不受AII受体拮抗剂的干扰。这些结果提示肾素-血管紧张素系统可能在中枢神经系统中发挥重要作用，不仅可以阻止All，还可以阻止AIV引起的神经元细胞凋亡。

The important research results in this project are summarized in the folowing two sections.1) Functional analysis of angiotensinogen-knockout miceGenetically altered mice lacking angiotensinogen (Agt-KO) showed an expected phenotype, such as marked hypotension, but unexpected ones including abnormal kidney morphology, reduced survival rates of newborns, and impaired blood-brain barrier function after cold injury. To examine whether disruption of the angiotensinogen gene is responsible for the observed phenotypes, we generated a line of mice heterozygous for the mouse angiotensinogen gene under the control of a mouse metallothionein-I promoter(MT-Agt) and crossmated transgenic mice with Agt-KO mice. The resulting mic (MT-Agt(+/-)/Agt(-/-) : MT-Agt/KO) produced the plasma level of angiotensin I comparable to that of wild-type mice, and the mutant phenotypes were rescued.2) New function of angiotensinogen knock-out miceThe morphological analysis in a congenic line of angiotensinogen knock-out mice (Ag-KO) revealed the decreased density in granular layer cells of hippocampus and cerebellum, suggesting neuronal cell of Ag-KO susceptible to apoptotic cell death. This phenomenon was further studied by culture of the hippocampal neurons with decreased concentration of serum. Ag-KO neuronal cells, which showed apoptosis by lower concentration of the serum within several hours, however, survived much longer in the presence of angiotensin II (All) and IV(AIV). This anti-apoptotic action was not interfered by AII receptor antagonists. These results suggest that the renin-angiotensin system could play a critical role in central nervous system, preventing neuronal cells from apoptosis not only by All but also AIV.