人端粒结合因子在端粒酶阳性和阴性细胞中的定位与周期性表达的研究

Localization of human telomere repeat binding factor 1 in telomerase positive and negative cells and its expression during cell cycle

目的 :研究人端粒结合因子 1(telomere repeatbinding factor 1,TRF1)在端粒酶阳性和阴性细胞中的定位以及在细胞周期中的表达。方法 :应用分子克隆技术扩增 TRF1基因全长 (TRF1FL )和 N、C端缺失突变体(TRF1ΔNC)序列 ,并克隆入 p EGFP- C2真核表达载体 ,表达绿色荧光 (GFP)融合蛋白 ;将含目的基因质粒转染端粒酶阳性 Hela细胞和端粒酶阴性 WI38- 2 RA细胞 ,Western Blot验证目的蛋白分子量 ,荧光显微镜观察 TRF1在间期细胞和染色体的定位 ;利用药物阻断 He La细胞于不同周期 ,流式细胞仪检测细胞周期 ,半定量 Western Blot检测 TRF1在不同细胞周期中的表达。结果 :TRF1FL、TRF1ΔNC序列长度分别为 1.3kb和 0 .9kb;GFP融合TRF1FL、TRF1ΔNC分子量大小分别为 80 k D和 6 0 k D。TRF1FL在间期细胞胞核内呈点状表达 ,在染色体则表达于染色体未端 ,TRF1ΔNC在细胞核内呈弥散性表达 ;TRF1在端粒酶阴性细胞中与早幼粒细胞白血病小体(promyelocytic leukemia body,PML )共定位 ,在端粒阳性细胞中则没有共定位。TRF1在 He La细胞中以 M期表达量最高 ,G1/ S表达最低 ,M期表达量是 G1/ S期的 3.9倍 (t=12 .92 ,P<0 .0 1)。结论 :TRF1在端粒酶阳性和阴性细胞中有不同的定位模式 ,在 He L a细胞中

Objective: To observe the distribution pattern of human telomere repeat binding factor 1(TRF1) in the telomerase positive (HeLa) and telomerase ngative cells (WI38 2RA) and to investigate its expression level during the cell cycle. Methods: The full length sequences of TRF1(TRF1FL) and its mutant with N and C terminus deletion (TRF1 Δ NC) were generated by PCR amplification,the resulting fragments were cloned into pEGFP C2 mammalian expression vector.GFP tagged proteins were verified by Western blotting with rabbit anti TRF1 and mouse anti GFP antibodies after cell transfection.Immunofluorescence staining were performed to detect the TRF1 localization in HeLa and WI38 2RA cells.Metaphase spreads from HeLa cells were also prepared to observe TRF1 localization in chromosomes.HeLa cells were arrested by thymidine and nocodazole at different cell stages.Cell cycles were analyzed by flow cytometry and TRF1 levels were evaluated by semi quantitative Western blotting. Results: TRF1FL and TRF1 Δ NC fragments were sized about 1.3 kb and 0.95 kb.GFP tagged TRF1FL and TRF1 Δ NC proteins were 80 kD and 60 kD, respectively.In both HeLa and WI38 2RA cells,TRF1FL had a speckled distribution in the nuclei,however,TRF1FL did not coincide with promyelocytic leukemia (PML) nuclear body in HeLa cells while it exclusively did in WI38 2RA cells.Moreover,TRF1FL was exactly localized at the termini of metaphase spreads in HeLa cells.In contrast,TRF1 Δ NC was diffusely distributed throughout the nuclei.Analysis by semi quantitative Western blotting indicated that TRF1 levels increased with cell cycle progression, which reached the zenith at the M phase and went down to the nadir at G 1/S point.The TRF1 level at M phase was about 3.9 times than that at G 1/S point( t =12.92, P <0.01). Conclusion: TRF1 has a different localization in telo merase positive and telomerase negative cells,which suggests TRF1 might exert different functions in these cells.TRF1 level is regulated with cell cycle. [