机械牵张诱导THP-1细胞高迁移率族蛋白B1移位出核

Mechanical stretch induces translocation of high-mobility group box 1 protein in THP-1 cells

目的:探讨机械牵张对高迁移率族蛋白B1(HMGB1)在THP-1细胞内移位的影响。方法:构建HMGB1-增强型绿色荧光蛋白(EGFP)融合蛋白的真核表达载体。通过两步亚克隆的方法,将HMGB1和EGFP的编码序列以融合蛋白的形式克隆到pcDNA3-HA载体上,随后转染THP-1细胞并施加机械牵张应变,荧光显微镜观察HMGB1在细胞内的表达及移位情况。结果:重组质粒经酶切鉴定证明构建正确,并在THP-1细胞中大量表达。荧光显微镜观察发现,HMGB1-EGFP融合蛋白主要分布于细胞核,经机械牵张18 h,可见细胞中融合蛋白从细胞核移位到细胞质,在细胞浆中观察到明显的绿色荧光。结论:成功构建了HMGB1-EGFP融合蛋白,在THP-1细胞中观察到机械牵张可诱导HMGB1移位出核。

AIM： To investigate the translocation of high - mobility group box 1 protein （ HMGB1 ） in THP - 1 ceils induced by mechanical stretch. METHODS： The vector that expressed the fusion protein of HMGB1 and enhanced green fluorescent protein（EGFP） was constructed. The cDNA of HMGB1 and EGFP was subcloned into hemagglutinin （HA） -tagged vector pcDNA3 -HA by two -step method. THP-1 ceils were transfected with pcDNA3 -HMGB1 -EG- FP and exposed to cyclic mechanical stretch at 20 % elongation using Flexereell 4000T cell stretching unit. The translocation of HMGB1 in THP - 1 cells was observed under fluorescence microscope. RESULTS ： The recombinant plasmid was verified by enzyme digestion. The green fluorescence accumulated in the nuclei of the cells, indicating that the fusion protein was highly expressed in THP - 1 cells and localized in the nuclei. Eighteen hours after mechanical stretch, the green fluorescence was observed in the cytoplasm. At the same time, the green fluorescence was still localized in the nuclei of the control cells treated without mechanical stretch. CONCLUSION： The HMGB1 -EGFP fusion protein is successfully and effectively expressed in THP - 1 cells. Mechanical stretch induces the translocation of HMGB1 protein from nucleus to cytoplasm.