NPM基因在人淋巴母细胞中的差异表达与染色体不稳定性

Correlation between Nucleophosmin Expression and Chromosome Instability in Human Lymphoblastoid Cells

目的:研究NPM基因与染色体不稳定性(CIN)、细胞增殖及p53的关系,为阐明恶性血液病发生的分子机制提供参考。方法:采用软琼脂克隆形成法、常规染色体分析和G显带法检测人淋巴母细胞TK6(wtp53)、WTK1(mtp53)和正常人淋巴细胞永生化细胞(HNILL)的细胞增殖、染色体数量变化和5号染色体质量排,并观察Olomoucine对其的影响;用Sanger测序法和Western Blot方法分别测定NPM第12外显子的DNA序列、NPM蛋白和磷酸化蛋白的表达。结果:WTK1细胞的克隆形成能力和多倍体率均显著高于TK6细胞,同时WTK1细胞的NPM蛋白和磷酸化蛋白表达亦明显高于TK6细胞,且它们均又显著高于HNILL细胞;3株细胞均未发现5号染色体的重排和NPM第12外显子编码区的突变。用Olomoucine抑制NPM蛋白磷酸化后WTK1细胞的多倍体率明显降低。结论:WTK1、TK6和HNILL细胞的增殖能力、CIN与NPM蛋白和磷酸化蛋白表达呈正相关,而且与p53状态密切相关。

Objective：To investigate the relationship between NPM and chromosome instability,cell proliferation and p53 and to elucidate the molecular mechanism of hematologic malignancies genesis. Methods：The cell proliferation, numerical chromosomal abnormalities and chromosome 5 rearrangements were assayed by soft agar colony formation test, conventional chromosome aberration analysis and G-band karyotype analysis in human lymphoblastoid cell lines TK6（wt p53）,WTK1 （mt p53）and human normal immortal lymphocyte line（HNILL）, respectively. The mutations of the 12^th exon of NPM and expression of NPM protein and phosphoprotein were detected by Sanger technology and Western Blot, respectively. The effects of olomoucine on numerical chromosomal abnormalities were observed in TK6 and WTK1 cells. Results： Colony-formation efficiency （CFE） and the percentage of polyploid cells of WTK1 cells were significantly higher than that of TK6 cells. Meanwhile expressions of NPM protein and phosphoprotein in WTK1 cells were markedly higher than that of TK6 cells. These effects of TK6 and WTKI cells were significantly higher than that of HNILL. No chromosome 5 translocations or mutations in the coding region of 12^th exon of NPM gene were observed in the three cell lines. Olomoucine could reduce the percentage of polyploid WTK1 cells. Conclusion：There were positive correlation between cell proliferation, CIN and NPM protein and phosphoprotein expression in WTK1 （mr p53）, TK6（wt p53） and HNILL cells, which also have close relationship with p53.