目的以自发性高血压大鼠(SHR)为研究对象,探讨盐负荷对心肌局部胶原成分、心肌细胞凋亡、血管紧张素Ⅱ受体AT1和AT2蛋白表达的影响。方法成年雄性SHR 60只,随机分为低盐(n=30)和高盐饮食组(n=30),分别用9g/L Na Cl和40g/L Na Cl干预3周。鼠尾法测量血压,尿钠测定;称体重,石蜡切片Mallory染色法观察心肌间质纤维化程度,TUNNEL法检测凋亡,免疫组化SABC法测定AT1和AT2蛋白表达。结果高盐负荷后SHR大鼠的血压、尿钠明显升高(P<0.05);左心室肌凋亡增多,胶原纤维沉积亦增多(P<0.05);盐负荷均能显著增加左心室AT1和AT2表达(P<0.01),AT1/AT2比值增大(P<0.05)。结论高盐负荷可导致SHR大鼠左心室肌重塑,心肌组织局部血管紧张素Ⅱ受体(ATR)的改变可能是SHR大鼠血管壁重塑的机制之一,减少盐的摄入对预防高血压引发的心肌肥厚、心衰的发生和发展有着重要的意义。
Objective： To construct a human ether-a-go-go-related gene （HERG） nonsense mutant L539fs/47-558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 （HEK293） cells. Methods： The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 1 and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-＊558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results： The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-＊558Wand the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery pEGFP-C2-L539fs/47＊-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. Conclusion： pEGFP-C2-L539fs/47-＊558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-＊558W
Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47.
