CAJ | 학술논문

目的研究胰岛素样生长因子结合蛋白-6(IGFBP-6)在细胞中的定位.方法用重叠PCR法构建IGFBP-6的核定位序列缺失体(pEGFP-C1-BP6ΔNLS)及突变体(pEGFP-C1-BP6-Mut)质粒,与野生型IGFBP-6质粒分别转染PC-3M细胞,激光扫描共聚焦显微镜观察绿色荧光融合蛋白在细胞中的分布,并计算这些转染阳性细胞中绿色荧光强度的核质比(Fn/c).结果激光扫描共聚焦显微镜观察显示,野生型IGFBP-6绿色荧光信号在细胞核内聚集分布,与转染EGFP空载体的对照组相比,Fn/c差异具有显著性(P<0.05).过表达pEGFP-C1-BP6ΔNLS的细胞中,绿色荧光信号在细胞核内聚集的现象消失,在整个细胞呈均匀分布;过表达pEGFP-C1-BP6-Mut的细胞中,细胞核内绿色荧光信号也有所减弱;两者的Fn/c与转染野生型IGFBP-6的细胞相比差异均具有显著性(P<0.05).结论 IGFBP-6可以定位于细胞核内,它在细胞核内的分布与其核定位序列相关,为进一步研究IGFBP-6不依赖于胰岛素样生长因子的生物学活性提供了新的实验依据.
목적 연구이도소양생장인자결합단백-6(IGFBP-6)재세포중적정위.방법 용중첩PCR법구건IGFBP-6적핵정위서렬결실체(pEGFP-C1-BP6ΔNLS)급돌변체(pEGFP-C1-BP6-Mut)질립,여야생형IGFBP-6질립분별전염PC-3M세포,격광소묘공취초현미경관찰록색형광융합단백재세포중적분포,병계산저사전염양성세포중록색형광강도적핵질비(Fn/c).결과 격광소묘공취초현미경관찰현시,야생형IGFBP-6록색형광신호재세포핵내취집분포,여전염EGFP공재체적대조조상비,Fn/c차이구유현저성(P<0.05).과표체pEGFP-C1-BP6ΔNLS적세포중,록색형광신호재세포핵내취집적현상소실,재정개세포정균균분포;과표체pEGFP-C1-BP6-Mut적세포중,세포핵내록색형광신호야유소감약;량자적Fn/c여전염야생형IGFBP-6적세포상비차이균구유현저성(P<0.05).결론 IGFBP-6가이정위우세포핵내,타재세포핵내적분포여기핵정위서렬상관,위진일보연구IGFBP-6불의뢰우이도소양생장인자적생물학활성제공료신적실험의거.
Objective To study the nuclear localization of insulin-like growth factor binding protein-6(IGFBP-6) in PC-3M cells. MethodsThe two fragments of the nuclear localization sequence (NLS)-deleted IGFBP-6 and the NLS-mutated IGFBP-6 were obtained by overlapping PCR, and then the fragment was inserted into a pEGFP-C1 vector. PC-3M cells were transfected with the expression constructs containing wild-type IGFBP-6 or the two mutants (pEGFP-C1-BP6ΔNLS and pEGFP-C1-BP6-Mut), and the different distribution of the three EGFP-fusion proteins was observed by confocal laser microscope. The statistical analysis of the ratio of the nuclear fluorescence to the cytoplasmic fluorescence (Fn/c) was performed. ResultsConfocal microscopic images of transfected cells showed that the green fluorescence of EGFP-IGFBP-6 was concentrated mostly in the nuclei, whereas the control cells expressing EGFP showed green fluorescence distributed uniformly. The results of Fn/c from EGFP and EGFP-IGFBP-6 were significant different (P<0.05). The NLS-deleted IGFBP-6 completely eliminated nuclear accumulation of the green fluorescent signal; in contrast, nuclear accumulation was only slightly reduced for the NLS-mutated IGFBP-6; compared with wild-type IGFBP-6, both mutants were significantly different (P<0.05). ConclusionsIGFBP-6 can be translocated to the nucleus in PC-3M cell that is mediated by a putative NLS sequence. Our study provides new evidence for further studies on the insulin-like growth factor-independent activity of IGFBP-6.