GFP-TET1过表达稳定细胞系的筛选及功能探讨  

Screening and Function of GFP-TET1 Overexpression Stable Cell Lines

作　　者：王瑶瑶 汪英男 潘巍巍[2] WANG Yao-yao;WANG Ying-nan;PAN Wei-wei(Wulanchabu Clinical Medical College,Baotou Medical College,Jining 012000 China;College of Medicine,Jiaxing University]raxing 314001 China)

机构地区：[1]包头医学院乌兰察布临床医学院,内蒙古集宁012000 [2]嘉兴学院医学院生物教研室,浙江嘉兴314001

出　　处：《内蒙古医学杂志》2020年第2期129-132,258,共4页Inner Mongolia Medical Journal

基　　金：国家自然科学基金项目(编号:NO81402162);内蒙古自治区卫生计生科研计划项目(编号:NO.201703236)。

摘　　要：目的本文通过构建带有绿色荧光蛋白的TET1过表达慢病毒载体,观察GFP-TET1在宫颈癌细胞中的亚细胞定位,初步探索TET1在宫颈癌细胞中的作用。方法以含TET1全长cDNA序列的质粒为模板,通过PCR扩增得到TET1基因,酶切连接后构建pLenti-GFP-TET1过表达慢病毒载体,琼脂糖凝胶电泳和测序验证后,通过包装、感染和嘌呤霉素筛选,获得稳定表达GFP-TET1的人宫颈癌CaSki细胞,通过Western blot检测TET1的表达情况,免疫荧光染色确定TET1亚细胞定位,细胞增殖曲线和软琼脂克隆形成实验检测TET1过表达对细胞增殖和细胞克隆形成能力影响;流式细胞检测TET1过表达细胞周期变化。结果酶切鉴定及测序结果均显示成功构建pLenti-GFP-TET1过表达的慢病毒载体,包装病毒后感染CaSki细胞获得TET1稳转细胞系;定量PCR和Western blot结果显示稳转细胞系中TET1 mRNA(P<0.001)和蛋白表达水平显著高于对照细胞;细胞增殖和克隆形成实验结果显示TET1过表达明显抑制细胞增殖(P<0.001)和克隆形成数目(P<0.01);免疫荧光染色结果表明TET1定位于整个细胞;细胞周期结果显示TET1过表达影响。结论我们成功的获得了pLenti-GFP-TET1稳定过表达的细胞系,并且初步探讨了过表达TET1在宫颈癌细胞中的作用。Objective To construct TET1 overexpression lentiviral plasmids with green fluorescent protein to observe the subcellular localization of GFP-TET1 in cervical cancer cells, and explored the effect of TET1 overexpression on cervical cancer cells.Methods The plasmids containing the full-length cDNA sequence of human TET1 were amplificated by PCR method. Human TET1 gene was digested and ligated to lentiviral pLenti-GFP-TET1 plasmid. The recombi nant vectors were confirmed by electrophoresis and sequencing. TET1 overexpression lentiviral vector was packaged lentiviral particles, then infected CaSki cells, cells were cultured in DMEM medium with puromycin(2 μg/ml) for 3 days and obtained stable expression of GFP-TET1 in human cervical cancer CaSki cells. Western blotting detected TET1 protein expression. Subcellular localization of TET1 protein was detected by immunofluorescence. Cell proliferation and clone formation were detected by MTT and soft agar assay. Cell cycle change of TET1 overexpression were detected by flow cytometry.Results The recombinant plasmid pLenti-GFP-TET1 was successfully established and confirmed by restriction endonuclease digestion and sequencing. pLenti-GFP-TET1 lentiviral virus was packaged and infected CaSki cells and obtained stable expression TET1 CaSki cells. The mRNA and protein level of TET1 were significantly increased in GFP-TET1 cells(P<0.001).TET1 overexpression inhibited cell colony formation(P<0.01).and cell proliferation(P<0.001).Immunofluorescence staining showed that TET1was located in the whole cell. FACS results showed TET1 overexpression affected cell cycle.Conclusion We successfully obtained a stable overexpressed pLenti-GFP-TET1 Caski cell and preliminarily explored the role of overexpressed TET1 in cervical cancer cells.

关 键 词：TET1 宫颈癌 细胞增殖 

分 类 号：R323.39[医药卫生—人体解剖和组织胚胎学]