【摘要】 生长抑素(SS)是一种多功能的脑肠肽,在动物生长轴中主要作为生长激素(GH)的负性调控因子。本研究通过慢病毒表达质粒介导shRNA,瞬时转染自身表达SS的BHK-21细胞,筛选出了靶向小鼠SS的最有效的siRNA序列。首先,分别在小鼠SS基因mRNA的246、433和539bp处找到潜在靶位点,合成3条siRNA转录模板的发夹结构以及1条阴性对照,体外退火后插入pshRNA-copGFP lentivector构建重组质粒。然后,通过对转染BHK-21细胞的荧光观察、Real-time PCR、免疫组化以及RIA等检测,转染细胞SS在mRNA水平及蛋白水平均检测到不同程度的抑制(P<0.05)。其中,psh2(SS 433～451)表现出了最高的沉默效率(转染效率68.9%时,mRNA水平的沉默效率达59.3%,P<0.05;蛋白质水平的沉默效率达55.6%,P<0.05)。本研究为降低SS在动物体内的分泌,相应提高GH的浓度,进而为促进动物生长的研究奠定了基础。更多还原

【Abstract】 As a multifunctional braingut petide,Somatostatin(SS) is an important negative regulator of Growth hormone(GH) in the GH-IGF-1 growth axis.In this study,we screened the best siRNA sequence targeting mouse SS by the transient-transfection of the lentiviral mediated shRNA into BHK-21 cells which secrete SS themselves.First,three potential sites in mouse SS mRNA sequence(start from 246,433,539 bp,seperately) were selected and synthesized,as well as one negative shRNA.After annealing in vitro,four of pshRNA-copGFP lentivectors were reconstructed.Then,the lentiviral-shRNAs were transient-transfected into BHK-21 cells,both SS mRNA and protein levels were down-regulated significantly(P<0.05) at 48 h post-transfection detected by the fluorescent observation,Real-time PCR,the immuno-histochemical analysis and RIA detection.Among which,sh2(SS 433-451) showed the highest silencing efficiency(SS mRNA reduced 59.3%,P<0.05 and SS protein reduced 55.6%,P<0.05 when the transfection efficiency was 68.9%).Our result laid a basis for the animal growth promotion via the down-regulation of SS.更多还原