阪崎肠杆菌培养上清蛋白引起HepG2肝细胞的基因表达变化

doi:10.3969/j.issn.1004-616x.2018.04.003

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (4): 258-262.doi: 10.3969/j.issn.1004-616x.2018.04.003

阪崎肠杆菌培养上清蛋白引起HepG2肝细胞的基因表达变化

由淑萍^1,4, 任立松², 马龙¹, 贺笋², 陈泽良², 王嵘³, 刘涛¹

1. 新疆医科大学公共卫生学院, 新疆乌鲁木齐 830011;
2. 新疆天康畜牧生物技术股份有限公司, 新疆乌鲁木齐 830032;
3. 中国解放军 69337部队, 新疆额敏 834601;
4. 新疆医科大学护理学院, 新疆乌鲁木齐 830011

收稿日期:2017-10-09 修回日期:2018-05-24 出版日期:2018-07-30 发布日期:2018-07-30
通讯作者: 刘涛,E-mail:xjmult@163.com E-mail:xjmult@163.com
作者简介:由淑萍,E-mail:youshupin@163.com
基金资助:
新疆维吾尔自治区自然科学基金（2014211C005）

Differential gene expression of supernatant proteins from Cronobacter sakazakii on cytopathy of HepG2 cells

YOU Shuping^1,4, REN Lisong², MA Long¹, HE Sun², CHEN Zeliang², WANG Rong³, LIU tao¹

1. College of Public Health, Xinjiang Medical University, Urumqi 830011;
2. Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd., Urumqi 830032;
3. Chinese People's Liberation Army 69337 Troops, Eminem 834601;
4. School of Nursing, Xinjiang Medical University, Urumqi 830011, Xinjiang, China

Received:2017-10-09 Revised:2018-05-24 Online:2018-07-30 Published:2018-07-30

摘要/Abstract

摘要： 目的：通过高通量测序法分析阪崎肠杆菌培养上清蛋白引起HepG2肝细胞的基因表达变化，并探讨阪崎肠杆菌致病作用的分子机制。方法：采用CAYE培养基培养阪崎肠杆菌，硫酸铵沉淀法及膜透析法分离纯化上清蛋白；分别在1×10⁶/mL的HepG2肝细胞培养液中加入终浓度为1.6 μg/mL的上清蛋白稀释液及蒸馏水作为实验组和对照组，提取细菌总RNA；构建文库并运用illumina测序平台测序，分析两组间的基因表达差异，采用Gene Ontology（GO）富集分析和Pathway分析了解差异表达基因的功能；荧光定量PCR（qPCR）验证RPS18、RFC4基因差异表达结果。结果：高通量测序结果显示，与对照组相比，实验组HepG2细胞存在5 120个差异表达基因，其中上调2 155个，下调2 965个；GO富集分析显示差异基因主要参与细胞核分裂、有丝分裂、复制等多个生物学过程，Pathway分析显示差异基因主要与DNA复制、蛋白转运、精氨酸和脯氨酸代谢等有关；qPCR验证结果与高通量测序结果一致。结论：阪崎肠杆菌培养上清蛋白能够引起HepG2肝细胞RPS18、RFC4等基因的表达变化，并可能与核糖体变化和/或DNA复制与修复有关。

关键词: 阪崎肠杆菌, 培养上清, HepG2肝细胞, 高通量测序

Abstract: OBJECTIVE: To investigate differential gene expression of supernatant proteins from Cronobacter sakazakii on cytopathy of HepG2 cells and to understand molecular interactions in pathopoiesis. METHODS: The CAYE culture medium was used to culture Enterobacter cloacae,and supernatant proteins were isolated and purified by the ammonium sulfate precipitation method. The supernatant proteins were added to the 1×10⁶/mL HepG2 hepatocyte culture at a final concentration of 1.6 μg/mL as the experimental group and distilled water was added as the control group. After 8 h of treatment,total RNA was extracted,the cDNA library was constructed and were sequenced using Illumina. The difference in gene expression between the two groups was analyzed. The function of differentially expressed genes was analyzed by the Gene Ontology (GO) enrichment analysis and Pathway analysis. Differential expression of RPS18 and RFC4 genes were validated using qPCR. RESULTS: High throughput sequencing results showed that there were 5 120 differentially expressed genes:2 155 were up-regulated and 2 965 were down-regulated. GO enrichment analyses showed that the differentially expressed genes were mainly involved in several biological processes,including mitosis,caryomitosis,replication,etc. Pathway analysis showed that the differentially expressed genes were mainly involved in DNA replication,protein transport,and arginine and proline metabolism. The results of qPCR and high-throughput sequencing were consistent. CONCLUSION: The supernatant proteins of Cronobacter sakazakii caused HepG2 liver cell damage which might be related to changes of ribosome and/or DNA replication,and repair with differential expression of RPS18 and RFC4 genes.

Key words: Cronobacter sakazakii, supernatant, HepG2 cell, high-throughput sequencing

中图分类号:

R155.5

由淑萍, 任立松, 马龙, 贺笋, 陈泽良, 王嵘, 刘涛. 阪崎肠杆菌培养上清蛋白引起HepG2肝细胞的基因表达变化[J]. 癌变·畸变·突变, 2018, 30(4): 258-262.

YOU Shuping, REN Lisong, MA Long, HE Sun, CHEN Zeliang, WANG Rong, LIU tao. Differential gene expression of supernatant proteins from Cronobacter sakazakii on cytopathy of HepG2 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(4): 258-262.

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阪崎肠杆菌培养上清蛋白引起HepG2肝细胞的基因表达变化

Differential gene expression of supernatant proteins from Cronobacter sakazakii on cytopathy of HepG2 cells

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