心钠素基因的突变及对其利尿活性的影响  

Site-directed mutagenesis of atrial natriuretic peptide gene and effect of the mutations on its diuretic activity in nephrotic rats

作　　者：李涛[1] 梁红雁[1] 闫峰[1] 卢圣栋[2] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所医学分子生物学国家重点实验室,北京100005 [2]中国医学科学院中国协和医科大学,北京100005

出　　处：《中华医学杂志》2002年第19期1324-1327,共4页National Medical Journal of China

基　　金：国家攀登计划资助项目 (93 0 2 110 0 3 )

摘　　要：目的　提高心钠素的生物学活性。方法　通过改造心钠素基因、构建突变型心钠素基因的逆转录病毒载体以及用肌内注射法将突变型和非突变型心钠素基因表达载体的裸DNA分别导入阿霉素诱导的肾病大鼠体内 ,以对其突变前后的利尿活性进行比较研究。结果　获得了结构和功能两方面均符合预期的突变型心钠素基因。与空载体组相比 [87± 7 1pg/ml(n =6 ) ],突变和野生型心钠素基因组大鼠 (n =6 )血浆中的心钠素浓度均在试验开始后的第 5d明显升高 ,分别为 10 7± 7 8pg/ml(t =4 6 5 ,P <0 0 1)和 113± 8 6pg/ml(t =5 71,P <0 0 1)。两个试验组大鼠的尿量在试验开始后的第 5d就明显增多 ,有效期达 15d以上。突变型心钠素基因的作用更强 ,其大鼠在第 5、10和 15d时的尿液增长量分别是野生型心钠素组的 1 6、2 0和 1 9倍。结论　所获突变型心钠素基因的利尿作用明显增强。Objective To prolong the half-life and enhance the biological activity of the human atrial natriuretic peptide (hANP), a peptide hormone, which is synthesized and released mainly by cardiac atrial myocytes and possesses potent natriuretic, diretic, and vasorelaxant properties Methods The site-directed mutation technique based on polymerase chain reaction was performed to get the mutant of the human ANP gene (mhANP), and the retroviral expression vector, pLHY24, in which mhANP gene is under the transcriptional control of the human cytomegalovirus promoter, was constructed The naked plasmid DNA of pLHY24 and positive control vector, pLHY19, in which the wild-type hNAP gene is in the same conditions as mhANP gene in pLHY24, and negative control vector, pLNCX without purpose gene, at a dose of 5 mg/kg body weight was injected intramuscularly into the rats with experimental renal disorder induced with adriamycin (ADR), respectively Results DNA sequencing result proved that the respected mhANP gene with the point mutations of TTC 131/Phe→TCC/Ser and ATG 135/Met→ATA/Ile has been obtained In comparison with negative control group (87 ±7 1 pg/ml), a single intramuscular injection of expression vector harboring mhANP or hANP gene resulted in an obvious increase in plasma level of mhANP (107 ±7 8 pg/ml, t = 4 65, P<0 01) or hANP (113 ±8 6 pg/ml, t = 5 71, P<0 01) 5 days after injection A significant elevation in the ratio of urine volume to body weight was occurred after both of mhANP gene and hANP gene delivery as compared with negative control and the effect lasted for more than 15 days The diuretic activity of mhANP gene delivery was 1 6-, 2 0-, and 1 9-fold higher than that of hANP gene 5, 10, and 15 days after gene transfer, respectively However, there were no statistical differences in the concentrations of K+ and Na+ in urine Conclusions Both of mhANP and hANP gene delivery into the rats with experimental nephropathy could improve their directic function obviously and the diuret

关 键 词：利尿活性 心钠素 基因疗法 逆转录病毒载体 心力衰竭 

分 类 号：R346[医药卫生—基础医学]