心脏特异表达肾素(原)受体转基因小鼠的建立

目的 建立心脏特异表达(p)RR的转基因小鼠,研究(p)RR在心肌病发病过程中的调节作用.方法 将(p) RR基因插入到心脏特异表达启动子α-MHC下游,构建转基因表达载体,通过显微注射的方法建立(p)RR转基因小鼠,PCR法鉴定转基因小鼠的基因型:Western blot和免疫组化的方法检测(p)RR在心脏组织中的表达；利用小动物超声仪检测转基因小鼠的心脏结构和功能；双色免疫荧光共聚焦进行(p)RR蛋白在转基因小鼠中的亚细胞定位,Western blot方法检测了钙泵(ATP2A2)、钠-钙交换体1(NCX 1)以及肌钙蛋白T(cTnT)在转基因小鼠心脏组织中表达量的变化情况.结果 建立了心脏高表达(p)RR的转基因小鼠品系；内源性和转基因高表达的(p)RR蛋白均定位在细胞内高尔基体和内质网上；心脏特异高表达(p)RR蛋白使小鼠心脏收缩功能增强,主要表现在与同窝阴性对照小鼠相比,转基因小鼠收缩期左室内径( LVID,systolic)降低9％ (P＜0.05,n=18),收缩期容积( LVESV,systolic)减少了23％ (P ＜0.05,n=18),射血分数EF( ejection fraction)升高14％ (P＜0.01,n=18),短轴缩短率FS( fraction shortening)升高20％ (P ＜0.01,n=18)；和心脏收缩功能和钙离子相关的ATP2A2和NCX 1表达均下调,而cTnT表达量上调.结论 在心脏中过表达(p)RR能够引起心脏收缩功能明显增强,同时直接或间接调节ATP2A2、NCX 1和cTnT表达.提示(p)RR可能是调节心脏中的钙离子而参与影响心肌功能.

Establishment of Heart-specific Overexpression of (pro) Renin Receptor Transgenic Mice

Objective To establish heart-specific(pro)renin receptor transgenic mice and investigate its regulation effect on the process of cardiomyopathy.Methods The transgenic vector was constructed by inserting the human(p)RR cDNA sequence into the down-stream of α-MHC promoter.The transgenic mice were generated by the method of microinjection.The genotype of transgenic lines was identified by PCR,and the expression levels of the(p)RR gene were detected by Western Blot and immunohistochemistry.The cardiac geometry and function were detected by echocardiography.Subcellular localization of(p)RR was performed by double immunofluorescence confocal staining.At last,we detected the expression level of ATP2A2,NCX1 and cTnT via Western Blot.Results The heart-specific(p)RR transgenic mice were established and one high-level expression line was identified.Endogenous and transgenic(p)RR were located in the Golgi body and endoplasmic reticulum.Overexpression(p)RR with heart-specific resulted in enhancing contractile properties.Compared with the wild type mice,transgenic mice left ventricular systolic diameter decreased by 9%(P 〈0.05,n=18),left ventricular end-systolic volume reduced by 23%(P 〈0.05,n=18),ejection fraction increased by 14%(P 〈0.01,n=18) and fraction shortening elevated by 20%(P 〈0.01,n=18).The expression level of the proteins involving in Ca2+ and contractile properties were altered,SRECA and NCX1 decreasing,while cTnT increasing.Conclusion Over-expression of(p)RR in the heart of the transgenic mice leaded to significantly enhanced cardiac contractile properties.,and while regulated the expression of SRECA,NCX1 and cTnT,directly or indirectly.These results indicated(p)RR participated in influencing cardiac function via regulating the Ca2+ of the myocardium.