SIRT2的亚细胞定位及其对293T细胞增殖的影响

Subcellular Localization of SIRT2 and its Effect on 293T Cell Proliferation

目的:构建SIRT2的真核表达载体,并检测其在人胚肾细胞株293T中的亚细胞定位以及对293T细胞增殖的影响。方法:以SIRT2-p IRES质粒为模板PCR扩增获得SIRT2基因目的片段,然后将其克隆到真核表达载体p EGFP-C2中,使目的基因能够和GFP融合表达;采用脂质体法转染293T细胞,在荧光显微镜下观察融合蛋白的亚细胞定位;用核浆分离和Western印迹方法检测融合蛋白的亚细胞定位,加入出核抑制剂细霉素B,进行同样的处理;采用MTT法检测SIRT2对293T细胞增殖的影响。结果:SIRT2主要分布在细胞质中,加入细霉素B后,在细胞质和细胞核中都有明显分布;SIRT2在细胞质中时抑制293T细胞的增殖,当其入核时抑制作用减弱。结论:SIRT2具有核质穿梭功能,它的亚细胞定位是个动态变化过程;SIRT2抑制细胞的增殖,抑制作用的强弱与其亚细胞定位有关。

Objective： To construct the recombinant eukaryotic expression vector SIRT2-pEGFP-C2 and investi- gate the subcellular localization of SIRT2 protein in 293T cell lines and the effect on cell proliferation of 293T. Methods： The SIRT2 gene full-length fragment was obtained from SIRT2-pIRES, then it was subcloned into the eukaryotic expression vector pEGFP-C2, making the target gene and GFP gene express simultaneously. The recom- binant plasmid was transfected into 293T cell lines by LipofectAMINE 2000. The subcellular localization of the fu- sion protein was monitored by autofluorescence microscopy. Use the reagent making cytoplasm and nucleus sepa- rate and Western blotting to detect the subcellular localization of the fusion protein. Add the nuclear export inhibi- tor leptomycin B and then perform the same processing. MTT assay was used to detect the effect of SIRT2 on 293T cell proliferation. Results： SIRT2 was mainly distributed in the cytoplasm. After adding leptomycin B, there was SIRT2 protein obviously both in the nucleus and cytoplasm. 5IRT2 suppress the cell proliferation when it was located in the cytoplasm. With entering the nucleus, the inhibition was decreased. Conclusion： SIRT2 may be a nucleocytoplasmic shuttle protein, and its subcellular localization is a dynamic process. Otherwise, SIRT2 inhibit cell proliferation and the strength of the inhibition may be related to its subcellular localization.