自发性高血压大鼠肌肉注射心钠素基因的降压作用

Reduction of Blood Pressure by Intramuscular Injection of Atrial Natriuretic Factor Gene in Hypertensive Rats

目的：研究骨骼肌注射重组的人心钠素（ＡＮＦ）基因对高血压动物的降压效应及机制。方法：用反转录酶链式反应（ＲＴ－ＰＣＲ）从人胚胎脑组织克隆出全长人ＡＮＦ互补脱氧核糖核酸（ｃＤＮＡ），并将其重组进质粒ｐｃＤＮＡ３，形成重组质粒ｐｃＤＮＡ３／ＡＮＦ。给５周龄（幼年）和１１周龄（成年）卒中易感型自发性高血压大鼠（ＳＨＲｓｐ）的股四头肌分别注入２００μｇ和６５０μｇｐｃＤＮＡ３／ＡＮＦ，并用放射免疫分析、ＲＴ－ＰＣＲ、Ｗｅｓｔｅｒｎ杂交和免疫组化技术检测人ＡＮＦｃＤＮＡ在体内的表达。每周测血压１次。最后在麻醉下收集尿和血，测定某些生化参数。结果：一次肌肉注射人ＡＮＦｃＤＮＡ可明显延缓幼年ＳＨＲｓｐ的血压升高；能降低成年ＳＨＲｓｐ的血压达２．７～４．０ｋＰａ（２０～３０ｍｍＨｇ），并伴有血浆ＡＮＦ浓度的明显升高及尿量、尿钠排泄的增加，局部肌肉组织的信使核糖核酸（ｍＲＮＡ）和ＡＮＦ表达阳性。结论：肌肉注射人ＡＮＦ基因可引起ＳＨＲｓｐ的持续性血压下降，显示了人ＡＮＦ基因对高血压患者降压治疗的可能性。

Objective:To investigate the hypotensive effect and mechanisms of the intramuscular injection of hANF cDNA in stroke prone spontaneously hypertensive rats(SHRsp). Methods:A full length hANF cDNA was amplified from human embryo brain by reverse transcription polymerase chain reaction(RT PCR).It was cloned into plasmid pcDNA3 to form the pcDNA3/ANF construct.Two hundred μg and 650 μg of pcDNA3/ANF was injected into the skeletal muscle of young(5 weeks) and adult(11 weeks) SHRsp,respectively.The caudal arterial pressure was measured once a week.Expression of hANF gene in the rats was measured by radioimmunoassay,RT PCR,western blot and immunohistochemistry and finally,the blood and urine were collected for determination of some biochemical parameters. Results:A single injection of naked hANF plasmid DNA caused a significant delay of hypertension in young SHRsp,while a 20—30 mmHg reduction of blood pressure in adults for at least 6 weeks as compared with controls injected with vector DNA alone ( p<0 01). After delivery of hANF cDNA,the expression of mRNA and ANF in the muscle was identified,and there were significant increases in plasma ANF,urinary volume and urinary sodi5m but not in plasma angiotensionⅡ,aldosterone, urinary potassium,creatinine,uric acid and urinary protein.The antibody to hANF was not detected in rat serum,either. Conclusion:The fact that the sustained reduction of systemic blood pressure in SHRsp induced by a single intramuscular injection of hANF cDNA suggests the feasibility of hANF gene therapy in human hypertension.