[1]黄小琴ab,涂名进,余华军ab,等.应用CRISPR/Cas 9 系统构建肺癌细胞系获得AMPKα1 基因敲除的稳定细胞株[J].现代检验医学杂志,2023,38(01):107-111.[doi:10.3969/j.issn.1671-7414.2023.01.020]
 HUANG Xiao-qinab,TU Ming-jin,YU Hua-junab,et al.Application of CRISPR/Cas 9 System to Construct Lung Cancer Cell Lines to Obtain Stable Cell Lines with AMPK α1 Knockdown[J].Journal of Modern Laboratory Medicine,2023,38(01):107-111.[doi:10.3969/j.issn.1671-7414.2023.01.020]
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应用CRISPR/Cas 9 系统构建肺癌细胞系获得AMPKα1 基因敲除的稳定细胞株()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年01期
页码:
107-111
栏目:
论著
出版日期:
2023-01-15

文章信息/Info

Title:
Application of CRISPR/Cas 9 System to Construct Lung Cancer Cell Lines to Obtain Stable Cell Lines with AMPK α1 Knockdown
文章编号:
1671-7414(2023)01-107-05
作者:
黄小琴12a2b涂名进3余华军2a2b贾玉芳12a2b严秀文12a2b汤喜莲12a2b郑健12a2b何柳燕3伍俊2a3张海涛12a2b
(1. 广东医科大学医学技术学院,广东东莞 523000;2. 广东医科大学a. 多肽和蛋白质研究与应用重点实验室;b. 生物化学与分子生物学系,广东湛江 524023;3. 广东医科大学附属医院呼吸内科,广东湛江 524023)
Author(s):
HUANG Xiao-qin12a2b TU Ming-jin3 YU Hua-jun2a2b JIA Yu-fang12a2bYAN Xiu-wen12a2b TANG Xi-lian12a2b ZHENG Jian12a2b HE Liu-yan3 WU Jun2a3 ZHANG Hai-tao12a2b
(1.School of Medical Technology, Guangdong Medical University,Guangdong Dongguan 523000,China;2a. Peptide and Protein Research and Application Key Laboratory;2b. Department of Biochemistry and Molecular Biology, Guangdong Medical University, Guangdong Zha
关键词:
CRISPR/Cas 9肺癌腺苷单磷酸活化蛋白激酶α1
分类号:
R734.2;R730.43
DOI:
10.3969/j.issn.1671-7414.2023.01.020
文献标志码:
A
摘要:
目的 利用CRISPR/Cas 9 系统在人肺癌细胞系A549 和H460 中获得敲除腺苷单磷酸活化蛋白激酶α1(adenosine monphosphate-activated protein kinase, AMPKα1) 的稳定表达细胞系。方法 根据CRISPR/Cas 9 靶点设计原则,设计三条引导RNA(sgRNA),分别构建AMPKα1-CRISPR/Cas9 KO 及其HDR 质粒。将质粒转染至A549 和H460 细胞后,筛选稳定的单克隆细胞株。用Western blot 鉴定筛选获得的肺癌细胞株是否表达AMPKα1。实验被分为原始对照组和敲除组,采用MTT 法检测AMPKα1 的肺癌细胞增殖情况。结果 经过CRISPR/Cas 9 系统处理后,筛选获得的A549 和H460 敲除AMPKα1 稳定细胞株与原始细胞株A549 和H460 相比,Western blot 检测不到AMPKα1蛋白质的表达,差异具有统计学意义(t=137.7, 79.17, 均P < 0.000 1)。MTT 结果显示,筛选获得的A549 和H460 敲除AMPKα1 的稳定细胞株与原始细胞株A549 和H460 相比,敲除细胞株的增殖速度明显减弱,差异具有统计学意义(t=3.956 ~ 28.16,均P < 0.05)。结论 CRISPR/Cas 9 系统成功建立敲除AMPKα1 表达的稳定肺癌细胞株,为进一步研究AMPKα1 在肺癌发生发展中的作用提供细胞模型。
Abstract:
Objective To construct stably lung cancer cell lines that did not express adenosine monphosphate-activated protein kinaseα1 (AMPKα1) using CRISPR/Cas 9 system. Methods According to the CRISPR/Cas 9 target design principle, three small guide RNA (sgRNAs) were designed to construct AMPKα1-CRISPR/Cas 9 KO and its HDR plasmid, respectively. After the plasmids were transfected into A549 and H460 cells, stable monoclonal cell lines were selected. The expression of AMPKα1 was detected in stable monoclonal cell lines by Western blot.The experiments were divided into original control and knockout groups, MTT assay was used to detect the proliferation of AMPKα1 knockout lung cancer cells. Results After treatment with the CRISPR/Cas 9 system, the knockdown AMPKα1 stable cell lines obtained from the screening were undetectable by Western blot for AMPKα1 protein expression compared with the original cell lines A549 and H460, and the differences were statistically significant (t=137.7, 79.17,all P < 0.000 1). The MTT results showed that the proliferation rate of knockdown cell lines obtained from the screened stable cell lines of A549 and H460 knockdown AMPKα1 was significantly weaker compared with the original cell lines A549 and H460 , and the differences were statistically significant (t=3.956 ~ 28.16, all P < 0.05).Conclusion The stable lung cancer cell lines with no expression of AMPKα1 were established by using CRISPR/CAS 9 system. It provides a cellular model for further studies on the role of AMPKα1 in lung carcinogenesis and development.

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备注/Memo

备注/Memo:
基金项目:广东医科大学学科建设项目(4SG21012G):麒麟菜多肽为有效成分研制防治IPF 药物的前期研究。
作者简介:黄小琴(1996-),女,硕士,检验技师,主要从事菜生化检验,E-mail:huangxiaoqin2333@163.com。
通讯作者: 伍俊(1973-),女,博士,主任医师,主要从事呼吸疾病研究,E-mail:wujun0294@163.com。
张海涛(1970-),男,博士,教授,主要从事肿瘤标志物研究,E-mail:taohaizhang@163.c
更新日期/Last Update: 2023-01-15