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目的最近研究表明核仁素(nucleolin,Ncl)蛋白作为一种内源性保护蛋白,在心肌细胞受损过程中发挥了重要的内源性保护作用。以往对核仁素发挥心肌保护作用的机制研究常局限于细胞水平。本研究构建并鉴定了携带有外源核仁素基因的转基因小鼠,为从整体水平上研究核仁素对心肌功能的影响,发挥心肌保护作用的机制提供动物模型。方法采用基因重组法将小鼠核仁素(Ncl)基因的全长cDNA置于α-肌球蛋白重链(α-MHC)启动子下游构建成α-MHC-Ncl重组载体,通过双酶切及基因测序鉴定其正确性;通过原核显微注射将该载体导入小鼠受精卵原核中,经注射且状态良好的受精卵回输至假孕雌鼠输卵管中,剪取0.5 cm仔鼠的鼠尾,收集DNA,进行PCR鉴定。结果双酶切及基因测序显示成功构建了α-MHC-Ncl重组质粒,将其显微注射到小鼠受精卵中,得到G0代小鼠106只,PCR确定了5只整合有Ncl基因的首建鼠。结论核仁素基因可以整合到小鼠体内,得到的转基因小鼠可能成为有效的动物模型,用于研究核仁素在心肌保护方面发挥的生物学作用。
Purpose Recent studies have shown that nucleoprotein (Ncl), as an endogenous protective protein, plays an important endogenous protective role in the process of cardiomyocyte injury. In the past, the mechanism of myocardial protection of nucleolin was often limited to the cellular level. In this study, transgenic mice carrying exogenous nucleolin gene were constructed and identified to provide an animal model for studying the effect of nucleolin on myocardial function and the mechanism of myocardial protection. Methods The full-length cDNA of mouse nucleolar renin (Ncl) gene was subcloned into the α-MHC-Ncl recombinant vector downstream of α-myosin heavy chain (α-MHC) promoter by gene recombination method. And gene sequencing to determine its correctness. The vector was introduced into the pronucleus of mouse fertilized egg by prokaryotic microinjection, and the fertilized ovum injected and in good condition was transfused into the fallopian tube of pseudopregnant female rats, Tail, collect DNA for PCR identification. Results Double enzyme digestion and gene sequencing showed that α-MHC-Ncl recombinant plasmid was successfully constructed and microinjected into mouse zygotes to obtain 106 G0 generation mice, and 5 heads of Ncl gene were identified by PCR Building mice. Conclusions Nucleotide genes can be integrated into mice and the resulting transgenic mice may be useful animal models for studying the biological role of nucleolin in cardioprotection.