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蛋白磷酸酶4(proteinphosphatase4,PP4)在果蝇、线虫及哺乳动物细胞中都可定位于中心体上,且在果蝇和线虫中参与了中心体成熟.为探讨PP4在哺乳动物细胞中的功能,通过RT-PCR的方法扩增得到PP4基因全长序列,构建pEGFP-C1-PP4融合蛋白表达质粒.将该质粒转染MCF7细胞,用间接免疫荧光技术确认PP4在MCF7细胞中的中心体定位.将PP4蛋白中非磷酸酶保守区序列作为反义抑制作用的靶序列,构建真核表达重组质粒pXJ41-as-PP4并转染MCF7细胞,G418筛选获得稳定的PP4表达受抑制的细胞株MCF7pXJ41-as-PP4.对此细胞株进行细胞形态、骨架结构、生长特性及有丝分裂过程观察分析,发现其生长速率明显减慢,血清依赖性增强,TdR双阻断进行周期同步化结合流式细胞术以及有丝分裂指数分析发现细胞进入M期受阻.间接免疫荧光检测发现,细胞中微管组织紊乱,细胞核物质增加,细胞群体中多核细胞比例增加,有丝分裂过程发生异常,出现较多多极分裂现象.这些结果表明,在哺乳动物细胞中,PP4的正常表达对中心体正常行使微管组织功能是必要的,抑制PP4表达将导致细胞增殖及周期进程发生异常.
Protein phosphatase 4 (PP4) is centrally located in Drosophila, nematode and mammalian cells and participates in centrosome maturation in Drosophila and Nematodes.To investigate the role of PP4 in mammalian cells The full-length sequence of PP4 gene was amplified by RT-PCR to construct the expression plasmid of pEGFP-C1-PP4 fusion protein.The plasmid was transfected into MCF7 cells and the central body localization of PP4 in MCF7 cells was confirmed by indirect immunofluorescence The non-phosphatase conserved region of PP4 protein was used as the target of antisense inhibition and the eukaryotic expression plasmid pXJ41-as-PP4 was constructed and transfected into MCF7 cells, and the stable cell line MCF7pXJ41 with suppressed PP4 expression was obtained by G418 screening -as-PP4. The cell morphology, skeletal structure, growth characteristics and mitotic process of this cell line observed and analyzed, found that the growth rate was significantly slowed, serum-dependent increase, TdR double block cycle synchronization with flow cytometry And mitotic index analysis found that the cells into the M phase obstruction.Indirect immunofluorescence test found that the cells in the microtubule tissue disorders, increased nuclear material, the proportion of cells in the multicellular nucleus increase Addition of mitotic processes resulted in more bipolar cleavage.These results suggest that the normal expression of PP4 in mammalian cells is essential for the normal functioning of centrosomes in microtubule organization and inhibition of PP4 expression will lead to cell proliferation and cycle Process anomalies.