基于CRISPR/Cas9的POR敲除THLE-2细胞模型及其在AFB1肝毒性研究中的应用

doi:10.7668/hbnxb.20196356

华北农学报 ›› 2026, Vol. 41 ›› Issue (2): 222-231. doi: 10.7668/hbnxb.20196356

• 畜牧·水产·兽医 • 上一篇下一篇

基于CRISPR/Cas9的POR敲除THLE-2细胞模型及其在AFB1肝毒性研究中的应用

于扬¹^,²^,³, 马玉晗¹^,²^,³, 李金峰¹^,²^,³, 陈嘉勇¹^,²^,³, 王丹¹^,²^,³, 陈玺竹¹^,²^,³, 余苗¹^,²^,³, 袁健¹^,²^,³, 娄佳楠¹^,²^,³, 曹三杰¹^,²^,³, 赵勤¹^,²^,³

¹ 四川农业大学动物医学院猪病研究中心, 四川成都 611130
² 农业农村部兽用药物与兽医诊断技术四川科学观测站, 四川成都 611130
³ 农业动物疫病与兽医公共卫生四川省重点实验室, 四川成都 611130

收稿日期:2025-08-09 出版日期:2026-05-06
通讯作者:
曹三杰(1971—),男,山西乡宁人,教授,博士,博士生导师,主要从事预防兽医学研究。
赵勤(1973—),女,四川成都人,副教授,博士,硕士生导师,主要从事预防兽医学研究。
作者简介:
于扬(2001—),女,四川绵阳人,在读硕士,主要从事预防兽医学研究。
基金资助:
川猪重大疫病防控新技术新产品创制(2021ZDZX0010); 国家级大学生创新训练项目(202310626006); 四川省“十四五”川猪重大科技专项

CRISPR/Cas9-Based POR Knockout THLE-2 Cell Model and Its Application in AFB1 Hepatotoxicity Research

YU Yang¹^,²^,³, MA Yuhan¹^,²^,³, LI Jinfeng¹^,²^,³, CHEN Jiayong¹^,²^,³, WANG Dan¹^,²^,³, CHEN Xizhu¹^,²^,³, YU Miao¹^,²^,³, YUAN Jian¹^,²^,³, LOU Jianan¹^,²^,³, CAO Sanjie¹^,²^,³, ZHAO Qin¹^,²^,³

¹ Research Center for Swine Diseases, College of Veterinary Medicine,Sichuan Agricultural University, Chengdu 611130, China
² Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Biotechnology, Ministry of Agriculture and Rural Affairs, Chengdu 611130, China
³ Agricultural Animal Diseases and Veterinary Public Health Key Laboratory of Sichuan Province, Chengdu 611130, China

Received:2025-08-09 Published:2026-05-06

摘要/Abstract

摘要：

前期通过CRISPR/Cas9文库筛选技术已鉴定出细胞色素P450氧化还原酶(POR)是促进黄曲霉毒素B1(AFB1)诱导肝细胞毒性的关键宿主因子。为进一步阐明POR蛋白在AFB1介导肝细胞损伤中的作用机制,基于已公布的人源POR基因序列,利用CRISPR/Cas9基因编辑技术,设计了2条靶向POR基因外显子5(exon 5)的sgRNA并克隆至lentiCRISPRv2载体,经慢病毒包装感染至THLE-2人肝细胞,用有限稀释法筛选单克隆细胞,并通过脱靶测序、RT-qPCR和Western Blotting在mRNA及蛋白水平验证敲除效率。结果表明:V2-sgRNA-POR-1可有效敲除POR基因,且筛出的单克隆细胞中POR蛋白的转录与翻译水平均显著下降。进一步采用不同浓度的AFB1(100,200,400,800 μmol/L)处理THLE-2野生型细胞24,48,72 h,CCK-8法细胞活力测定结果显示,AFB1对肝细胞的毒性具有明显的浓度和时间依赖性,其中200 μmol/L处理48 h为最适染毒条件[IC₅₀(48 h)=(200.55±44.49)μmol/L]。在相同暴露条件下,POR敲除细胞株对AFB1诱导的细胞死亡表现出显著的耐受性,细胞活力明显高于野生型细胞。综上,POR蛋白在AFB1介导的肝细胞毒性过程中发挥关键促进作用,敲除POR可显著增强细胞对AFB1的抵抗能力。

关键词: 黄曲霉毒素B1(AFB1), 细胞色素P450氧化还原酶(POR), CRISPR/Cas9, THLE-2细胞

Abstract:

In previous research,cytochrome P450 oxidoreductase(POR)was identified as a critical host factor facilitating aflatoxin B1(AFB1)-induced hepatotoxicity through CRISPR/Cas9 library screening.To further elucidate the mechanistic role of POR in AFB1-mediated hepatocellular damage,we designed two single-guide RNAs(sgRNAs)targeting exon 5 of the human POR gene,based on its published sequence,using CRISPR/Cas9 gene-editing technology.These sgRNAs were cloned into the lentiCRISPRv2 vector,which was subsequently packaged into lentivirus and utilized to infect THLE-2 human hepatocytes.Monoclonal cell lines were isolated by limiting dilution,and knockout efficiency was assessed at both the mRNA and protein levels using off-target sequencing,reverse transcription quantitative PCR(RT-qPCR),and Western Blot analysis.The findings indicated that V2-sgRNA-POR-1 successfully knocked down POR expression,resulting in a significant reduction at both the transcriptional and translational levels in the selected monoclonal cells.Further experiments involved treating THLE-2 wild-type cells with varying concentrations of AFB1(100,200,400,800 μmol/L)for durations of 24,48,and 72 hours.CCK-8 cell viability assays demonstrated that AFB1 toxicity to hepatocytes was significantly dependent on both concentration and exposure time,with an optimal exposure condition identified at 200 μmol/L for 48 hours[IC₅₀(48 h)=(200.55±44.49)μmol/L].Under these conditions,POR knockout cell lines exhibited marked resistance to AFB1-induced cytotoxicity,with significantly higher cell viability than wild-type cells.These findings suggest that POR plays a critical role in facilitating AFB1-mediated hepatocyte toxicity,and that POR knockout substantially enhances cellular resistance to AFB1.

Key words: Aflatoxin B1(AFB1), Cytochrome P450 oxidoreductase(POR), CRISPR/Cas9, THLE-2 cell

中图分类号:

S852.6

于扬, 马玉晗, 李金峰, 陈嘉勇, 王丹, 陈玺竹, 余苗, 袁健, 娄佳楠, 曹三杰, 赵勤. 基于CRISPR/Cas9的POR敲除THLE-2细胞模型及其在AFB1肝毒性研究中的应用[J]. 华北农学报, 2026, 41(2): 222-231. doi: 10.7668/hbnxb.20196356.

YU Yang, MA Yuhan, LI Jinfeng, CHEN Jiayong, WANG Dan, CHEN Xizhu, YU Miao, YUAN Jian, LOU Jianan, CAO Sanjie, ZHAO Qin. CRISPR/Cas9-Based POR Knockout THLE-2 Cell Model and Its Application in AFB1 Hepatotoxicity Research[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 222-231. doi: 10.7668/hbnxb.20196356.

http://www.hbnxb.net/CN/Y2026/V41/I2/222

图/表 8

表1 sgRNA-POR序列

Tab.1 Sequences for sgRNA-POR

靶位点 Target site	靶位点序列(5'—3') Target site sequence(5'—3')	引物序列(5'—3') Primer sequence (5'—3')
sgRNA-POR-1	AGAGATCGACAACGCCCTGG	F: ACCGAGAGATCGACAACGCCCTGG
		R: AAACCCAGGGCGTTGTCGATCTCTC
sgRNA-POR-2	GCCAGAGATCGACAACGCCC	F: GACCGCCAGAGATCGACAACGCCC
		R: AAACGGGCGTTGTCGATCTCTGGC

表2 POR基因敲除鉴定引物序列

Tab.2 POR gene deletion identification primer sequence

靶位点 Target site	引物序列(5'—3') Primer sequence (5'—3')
sgRNA-POR-1	F:AACCTTGAACAGGCTCAGTCAT
	R:ATACATGGATCTGGAGTCCCC
sgRNA-POR-2	F:AGCCCCTCCGTGTTGTTA
	R: AGGGGACATTCTCGTAGTGC

表3 RT-qRCR引物序列

Tab.3 Primer sequences for RT-qRCR

基因名称 Gene	序列号 Accession No.	引物序列(5'—3') Primer sequence (5'—3')	扩增子/bp Amplicon	退火温度/℃ Tm
POR	NM_000941.3	F: TCTCCTAACCTACTGGTTCCTCT	182	60.5
		R: TCCTCCCCGTTTTCTTCATCT
GAPDH	NM_002046.7	F: GGAGCGAGATCCCTCCAAAAT	197	60.0
		R: GGCTGTTGTCATACTTCTCATGG

图1 lentiCRISPR-v2-sgRNA重组质粒的构建 A.sgRNA-POR靶位点示意图;B.LentiCRISPR-V2-sgRNA-POR重组质粒模式图;C.重组质粒靶位点序列比对。

Fig.1 Construction of the lentiCRISPR-v2-sgRNA recombinant plasmid A.sgRNA-POR target site diagram;B.The model of LentiCRISPR-V2-sgRNA-POR recombinant plasmid;C.The sequence alignment results of the target sites of the recombinant plasmid.

图2 POR-KO单克隆细胞株的鉴定 A.梯度浓度的嘌呤霉素筛选浓度;B.POR基因敲除THLE-2细胞系测序结果;C.RT-qRCR结果;D.Western Blotting鉴定结果;E.Western Blotting灰度分析。不同小写字母表示组内差异达到显著水平P<0.05。

Fig.2 Identification of POR-KO monoclonal cell lines A.Screening of purine toxin concentrations;B.Sanger sequencing results of POR gene knockout THLE-2 cell lines;C.RT-qPCR results;D.Western Blotting analysis;E.Grayscale quantification of Western Blotting results.Different lowercase letters indicate statistically significant differences at P<0.05.

图3 THLE-2-WT细胞在AFB1暴露下的细胞存活率

Fig.3 Assessment of cell viability in THLE-2-WT cells following exposure to AFB1

图4 AFB1染毒暴露48 h后细胞形态观察(100×)

Fig.4 Morphological observation of cells after 48 h exposure to AFB1(100×)

图5 AFB1染毒暴露48 h后细胞存活率不同小写字母表示同一浓度染毒处理条件的组内差异达到显著水平P<0.05。

Fig.5 Cell survival rate after 48 h exposure to AFB1 Within the same treatment concentration,groups sharing the same lowercase letter are not significantly different at P<0.05.

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基于CRISPR/Cas9的POR敲除THLE-2细胞模型及其在AFB1肝毒性研究中的应用

CRISPR/Cas9-Based POR Knockout THLE-2 Cell Model and Its Application in AFB1 Hepatotoxicity Research

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