【摘要】 目的:建立人热休克蛋白27(heat shock protein27,Hsp27)基因在小鼠心肌特异性表达的转基因鼠模型。方法:将人心肌Hsp27cDNA插入含有心肌特异性表达启动子αMHC的pBSⅡ-SK+载体中,限制性内切酶EcoRI酶切、纯化后,获得含有αMHC启动子-Hsp27cDNA-HGHpolyA的线性DNA片段,以显微注射法将目的基因导入受精卵,PCR筛选基因型,Western blot鉴定转移基因的表达和表达的组织特异性。结果和结论:共获得两个转基因系小鼠,均呈心肌组织特异性高表达。更多还原

【Abstract】 Objective:Increased reactive oxygen species (ROS) formation and by which in turn promotes cardiomyocytes apoptosis is associated with the pathogenesis and progression of various cardiac diseases. Small heat shock protein 27(Hsp27) could protect different cells from oxidative damage. By using tissue nonspecific overexpression Hsp 27 transgenic model, other investigators demonstrated that Hsp27 suppressed successfully kainate-induced seizures and hippocampal cell death in intact transgenic mice, and attenuated mimic ischemia/reperfusion injury in Langendorff-perfused isolated mice heart. As there are complicated and long distance neuro-humoral regulation associated with the development of cardiac diseases, it is better to choose a cardiac-specific overexpression transgenic model to study the effects of Hsp27 in hearts in vivo.Methods:A cDNA encoding human Hsp27 was subcloned into pBSII-SK+ containing the α-myosin heavy chain (α-MHC) promoter (generously provided by Dr. J. Robbins, Children’s Hospital of Cincinnati, Ohio). BamHI-digested linear transgene consistent with the α-MHC promoter, Hsp27 cDNA, and poly (A) of human growth hormone (hGH) was microinjected into the fertilized eggs from CBA/BL6 mice. Mice containing the transgene were identified by polymerase chain reaction. Founders revealed by this screening were used to establish independent transgenic lines. Following successful transmission, a range of tissues including heart, lung, liver, brain, skeleton muscle, spleen and kidney was screened by Western blot to confirm the cardiac specific expression of the transgene. Results and Discussion:Transgenic mice expressed Hsp27 under the control of a-MHC promoter. Cardiac tissues from independent TG line expressed abundant Hsp27, and as expected, Hsp27 expressed in cardiac tissues only, whereas none in liver, spleen, lung, kidney, brain and skeleton muscle. Surprisingly, Hsp25, the endogenous isoform of Hsp27 in murine, was downregulated by Hsp27 overexpression (data not shown), which is in contrast to the result of Hollander’s [1]. It needs to determine in future whether Hsp27 expression profile in heart could exert any effect on the regulation of Hsp25.Conclusion: The TG mice overexpression human Hsp27 specifically in the heart by using αMHC- promotor were created. All TG mice expressed Hsp27 abundantly and cardiac-specifically. To our knowledge, it is the first report of creation of transgenic mice which overexpressing Hsp27 cardiac specifically. This current model suggests that the Hsp27 cardiac-specific over-expression of transgenic mouse remains a robust genetic tool for dissecting molecular and genetic events involving Hsp27, which could be a therapeutic target in heart failure.更多还原