核仁磷酸蛋白基因NPM1敲除人宫颈癌细胞株的构建及功能  

Construction and function of Nucleophosmin 1 gene(NPM1) knockout in human cervical cancer cells

作　　者：王昊然[1] 白晓[1] 闫丽 任燕红 孙威[1] 商维昊 WANG Hao-ran;BAI Xiao;YAN Li;REN Yan-hong;SUN Wei;SHANG Wei-hao(College of Life Sciences, Epigenetic Teaching and Research Department, China Medical University, Shenyang 110013, China)

机构地区：[1]中国医科大学生命科学院表观遗传教研室

出　　处：《解剖科学进展》2019年第4期353-356,360,共5页Progress of Anatomical Sciences

基　　金：国家自然科学基金(31771502)

摘　　要：目的通过CRISPR/Cas9系统定向敲除人类宫颈癌细胞(HELA)的核仁磷酸蛋白1基因(nucleophosmin1,NPM1),探讨敲除对HELA细胞生物学行为的影响。方法利用http://crispr.mit.edusgRNA在线设计工具设计sgRNA,通过T7E1系统筛选合适的sgRNA,将设计好的质粒通过转染的方式转入HELA细胞株,通过博来霉素(zeocin)抗生素进行筛选,通过Westernblot,PCR,基因测序等手段确认敲除细胞系的建立,通过免疫荧光的方式观察敲除细胞株。结果T7E1酶切PCR退火产物之后琼脂糖凝胶结果表明,CRISPR/Cas9系统对HELA细胞基因组进行了切割,并在同源重组时发生了碱基错配;Westernblot结果说明基因敲除后NPM1蛋白表达消失;基因测序的结果表明敲除细胞系建立的成功;免疫荧光的结果表明该细胞系出现异常核仁现象。结论NPM1敲除后细胞核仁异常,可能影响细胞正常生命进程。Objective To knock out the NPM1 gene in HELA cells by CRISPR/Cas9 system and to explore the effects of knockout on the biological behavior of HELA cells. Methods The sgRNA was designed using the online design tool of http://crispr.mit.edu sgRNA, and the appropriate sgRNA was screened by T7E1 system. The designed plasmid was transfected into HELA cell line and screened by zeocin antibiotic. Western blot, PCR, gene sequencing were used to confirm the establishment of knockout cell lines, and knockout cell lines were observed by immunofluorescence. Results The CRISPR/Cas9 system cleaves the genome of HELA cells and base mismatches occur during homologous recombination by agarose gel digestion and T7E1 digestion. The gene knockout NPM1 protein expression disappeared by Western blot immunoblot. The knockout cell line was established successfully by gene sequencing. The abnormal nucleoli phenomenon was found in the cell line by immunofluorescence. Conclusion The abnormal nucleolus of NPM1 knockout may affect the normal life course of cells.

关 键 词：核仁磷酸蛋白1 人类宫颈癌细胞 基因敲除 细胞增殖 

分 类 号：R737.33[医药卫生—肿瘤]