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通过CRISPR/Cas9系统建立α-缺陷型HepG2细胞系及其对复制影响的评估

Establishment of a -Deficient HepG2 Cell Line through CRISPR/Cas9 System and Evaluation of Its Effects on Replication.

作者信息

Sun Lanqing, Huang Kai, Huang Xuan

机构信息

Department of Laboratory Medicine Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China.

Orthopaedic Institute Wuxi Ninth People's Hospital Affiliated to Soochow University, Wuxi, Jiangsu, China.

出版信息

J Pathog. 2024 Sep 12;2024:9615181. doi: 10.1155/2024/9615181. eCollection 2024.

DOI:10.1155/2024/9615181
PMID:39301082
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11412752/
Abstract

BACKGROUND

serovar Typhimurium ( Typhimurium) is a common food-borne pathogen that causes gastroenteritis and can lead to life-threatening systemic disease when it spreads to vital organs, such as the liver. Stimulator of interferon genes (STING) is a crucial regulator of the host's innate immune response to viral infections, while its role in bacterial infections remains controversial. This study aims to establish a -deficient HepG2 cell line through the CRISPR/Cas9 system and evaluate its effects on replication.

METHODS

In this study, a knockout HepG2 cell line was constructed through the application of CRISPR/Cas9 technology. We assessed cell viability and proliferation using the CCK-8 assay. Subsequently, we investigated the effect of deletion on replication and the expression of type I interferon-related genes.

RESULTS

The knockout HepG2 cell line was successfully constructed using the CRISPR/Cas9 system. The proliferation capability was diminished in -deficient HepG2 cells, while Typhimurium replication in these cells was augmented compared to the wild-type (WT) group. Following infection, the transcriptional responses of type I interferon-related genes, such as and , were inhibited in -deficient HepG2 cells.

CONCLUSIONS

We successfully constructed a -deficient cell line. Our finding of increased Typhimurium replication in -deficient HepG2 cells provides the basis for further studies on pathogen-host interactions.

摘要

背景

鼠伤寒血清型（鼠伤寒）是一种常见的食源性病原体，可引起肠胃炎，当它扩散到重要器官（如肝脏）时可导致危及生命的全身性疾病。干扰素基因刺激物（STING）是宿主对病毒感染的固有免疫反应的关键调节因子，而其在细菌感染中的作用仍存在争议。本研究旨在通过CRISPR/Cas9系统建立STING缺陷的HepG2细胞系，并评估其对鼠伤寒复制的影响。

方法

在本研究中，通过应用CRISPR/Cas9技术构建了STING敲除的HepG2细胞系。我们使用CCK-8测定法评估细胞活力和增殖。随后，我们研究了STING缺失对鼠伤寒复制和I型干扰素相关基因表达的影响。

结果

使用CRISPR/Cas9系统成功构建了STING敲除的HepG2细胞系。STING缺陷的HepG2细胞的增殖能力降低，而与野生型（WT）组相比，这些细胞中鼠伤寒的复制增加。在感染鼠伤寒后，STING缺陷的HepG2细胞中I型干扰素相关基因（如IFN-β和IFN-α）的转录反应受到抑制。

结论

我们成功构建了STING缺陷细胞系。我们发现STING缺陷的HepG2细胞中鼠伤寒复制增加，这为进一步研究病原体-宿主相互作用提供了基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/99defe7191e5/JPATH2024-9615181.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/b29b95e8eb1a/JPATH2024-9615181.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/64256c8a305e/JPATH2024-9615181.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/f00304401a75/JPATH2024-9615181.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/99defe7191e5/JPATH2024-9615181.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/b29b95e8eb1a/JPATH2024-9615181.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/64256c8a305e/JPATH2024-9615181.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/f00304401a75/JPATH2024-9615181.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5e70/11412752/99defe7191e5/JPATH2024-9615181.004.jpg

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沙门氏菌通过触发线粒体 DNA 释放诱导小鼠巨噬细胞中的 cGAS-STING 依赖性 I 型干扰素反应。
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