论文部分内容阅读
TBP-like protein(TLP)是真核细胞中一种常见的转录因子,在调节生长发育方面起着重要的作用。该实验构建重组质粒pEGFP-N1-TLP,研究TLP对人宫颈癌细胞HeLa增殖的影响。利用流式细胞仪检测质粒的转染效率,通过激光共聚焦显微镜观察外源TLP蛋白的亚细胞定位。经过MTT检测、RNAi-TLP诱导的基因沉默及Hoechst33258染色研究TLP对HeLa细胞的增殖抑制作用。流式细胞术、Western blot和RT-PCR实验结果表明,TLP将HeLa细胞周期阻滞于G2/M期,并抑制周期相关基因CDK1和CyclinB1的转录和翻译。研究表明,外源TLP在HeLa细胞的细胞核中表达,通过降低细胞周期相关基因CDK1和CDK1的表达水平,将HeLa细胞的细胞周期阻滞于G2/M期,从而抑制细胞的增殖。
TBP-like protein (TLP) is a common transcription factor in eukaryotic cells and plays an important role in regulating growth and development. In this experiment, a recombinant plasmid pEGFP-N1-TLP was constructed to study the effect of TLP on the proliferation of human cervical cancer cell line HeLa. The transfection efficiency of plasmid was detected by flow cytometry. The subcellular localization of exogenous TLP protein was observed by laser confocal microscopy. MTT assay, RNAi-TLP-induced gene silencing and Hoechst33258 staining TLR on HeLa cell proliferation inhibition. Flow cytometry, Western blot and RT-PCR results showed that TLP blocked HeLa cell cycle in G2 / M phase and inhibited the transcription and translation of cyclinB1 and cyclinB1. Studies have shown that exogenous TLP is expressed in the nucleus of HeLa cells, which can inhibit the proliferation of HeLa cells by arresting the cell cycle of G2 / M phase in HeLa cells by decreasing the expression of CDK1 and CDK1.