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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 TRF1呈周期性表达
Objective: To study the targeting telomere binding factor 1 (telomere repeatbinding factor 1, TRF1) in telomerase positive and negative cells and expressed in the cell cycle. Methods: TRF1FL and TRF1ΔNC were amplified by molecular cloning technique and cloned into pEGFP-C2 eukaryotic expression vector to express green fluorescent protein (GFP) fusion protein. The target gene plasmids were transfected into telomerase-positive Hela cells and telomerase-negative WI38-2 RA cells. The molecular weight of the target protein was verified by Western Blot. The localization of TRF1 in interphase cells and chromosomes was observed by fluorescence microscopy. He La cells At different cycles, the cell cycle was detected by flow cytometry, and the semi-quantitative Western Blot was used to detect the expression of TRF1 in different cell cycle. Results: The lengths of TRF1FL and TRF1ΔNC were 1.3kb and 0.9kb, respectively. The molecular weights of TRF1FL and TRF1ΔNC were 80 kD and 60 kD, respectively. TRF1FL was spotted in the interphase nucleus, expressed in the chromosome at the end of the chromosome, TRF1ΔNC was diffusely expressed in the nucleus; TRF1 was expressed in the telomerase negative cells with the promyelocytic leukemia body, PML) colocalization, there is no co-localization in telomere positive cells. The expression of TRF1 was the highest in M phase and the lowest in G1 / S phase in HeLa cells. The expression of TRF1 was 3.9 folds of G1 / S phase (t = 12.92, P <0.01). CONCLUSIONS: TRF1 has different localization patterns in telomerase positive and negative cells, and TRF1 is expressed periodically in HeLa cells