脓毒性急性肾损伤小鼠线粒体DNA损伤修复相关基因的筛选

doi:10.3785/j.issn.1008-9292.2018.02.06

浙江大学学报（医学版）

2018, Vol. 47

Issue (1): 41-50 DOI: 10.3785/j.issn.1008-9292.2018.02.06

原著

脓毒性急性肾损伤小鼠线粒体DNA损伤修复相关基因的筛选

杨静娟(

),吴峰峰,陈江华,杨毅*(

)

浙江大学医学院附属第一医院肾脏病中心, 浙江杭州 310003

Screening of mitochondrial DNA damage repair genes in rats with septic acute kidney injury

YANG Jingjuan(

),WU Fengfeng,CHEN Jianghua,YANG Yi*(

)

Kidney Disease Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China

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摘要：

目的: 筛选脓毒性急性肾损伤（SAKI）中参与线粒体DNA（mtDNA）损伤修复的相关基因。方法: 40只清洁级雄性C57BL/6J小鼠随机分为SAKI组（28只）和对照组（12只）。采用盲肠结扎穿刺术建立SAKI小鼠模型。分别于术后8、24、48 h采集两组的血液和肾脏标本，干式生化仪检测血清肌酐和尿素氮，ELISA法检测血清炎症因子表达水平；HE染色观察肾脏组织病理学变化。RNA测序和生物信息学分析筛选mtDNA损伤修复相关基因；实时定量RT-PCR法和免疫组织化学法检测相关基因的mRNA和蛋白表达水平。结果: SAKI组小鼠术后均出现脓毒症症状，死亡16只；血清炎症因子（TNF-α和IL-6）、肌酐和尿素氮水平均高于对照组（P < 0.05或P < 0.01）；肾小管上皮细胞肿胀，炎症细胞浸润，并可见大量细胞空泡形成，提示建模成功。生物信息学分析筛选出Gadd45α、Bcl2l1、Cdkn1a、Jun、Rela、Nfkbia和Nfkb1等线粒体DNA损伤修复相关基因，实时定量RT-PCR检测以上基因表达量结果与RNA测序趋势一致。SAKI组Gadd45α主要在肾小管上皮细胞核中表达，其阳性表达率高于对照组（P < 0.05）。结论: Gadd45α、Bcl2l1、Cdkn1a、Jun、Rela、Nfkbia和Nfkb1基因参与了SAKI中mtDNA损伤修复，可以作为SAKI防治的新靶点。

关键词： 脓毒症; DNA损伤; 基因, 肿瘤抑制; 线粒体; 肾/损伤; 肾/病理学; 肾功能不全, 急性; 疾病模型, 动物

Abstract:

Objective: To screen genes involved in mitochondrial DNA (mtDNA) damage repair in rats with septic acute kidney injury (SAKI). Methods: Forty male C57BL/6J mice were randomly divided into SAKI group (n=28) and sham operation group (n=12). The SAKI mouse model was established by cecal ligation and puncture. Blood and kidney samples were collected at 8, 24, and 48 h after surgery. Serum creatinine and urea nitrogen were measured by a dry biochemical analyzer. Serum inflammatory cytokines were detected by ELISA. Histopathological changes were observed with HE staining. The mtDNA damage repair related genes were screened by RNA sequencing and bioinformatics analysis; the mRNA and protein expression levels of related genes were detected by real-time quantitative RT-PCR and immunohistochemisry, respectively. Results: Symptoms of sepsis were observed in SAKI group, and 16 out of 28 mice were died in the SAKI group; serum TNF-α, IL-6, creatinine and urea nitrogen levels were higher than those in the sham group (P < 0.05 or P < 0.01). Histopathological examination in SAKI group showed that renal tubular epithelial cells were swollen, inflammatory cells infiltrated, and a large number of cell vacuoles were seen, suggesting successful modeling. Mitochondrial DNA damage repair related genes Gadd45α, Bcl2l1, Cdkn1a, Jun, Rela, Nfkbia and Nfkb1 were screened out. The expression of these genes was detected by real-time RT-PCR, and the results were consistent with RNA sequencing trends. Immunohistochemical staining showed that Gadd45α was mainly expressed in the nucleus of renal tubular epithelial cells, and the positive rate of Gadd45α in SAKI group was higher than that in the sham operation group (P < 0.05). Conclusion: Gadd45α, Bcl2l1, Cdkn1a, Jun, Rela, Nfkbia and Nfkb1 genes are involved in mtDNA damage repair in rats with SAKI, indicating that these genes may be used as new targets for prevention and treatment of SAKI.

Key words: Sepsis DNA damage Genes, tumor suppressor Mitochondria Kidney/injuries Kidney/pathology Renal insufficiency, acute Disease models, animal

收稿日期: 2017-09-28 出版日期: 2018-06-12

CLC:

R36

基金资助: 国家自然科学基金(81670621);浙江省自然科学基金(LY16H050001)

通讯作者: 杨毅 E-mail: jingjuyang@126.com;yangyixk@zju.edu.cn

作者简介: 杨静娟(1993-), 女, 硕士研究生, 主要从事脓毒症和肾脏疾病的研究; E-mail:jingjuyang@126.com; https://orcid.org/0000-0002-4671-2519

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引用本文:

杨静娟,吴峰峰,陈江华,杨毅. 脓毒性急性肾损伤小鼠线粒体DNA损伤修复相关基因的筛选[J]. 浙江大学学报（医学版）, 2018, 47(1): 41-50.

YANG Jingjuan,WU Fengfeng,CHEN Jianghua,YANG Yi. Screening of mitochondrial DNA damage repair genes in rats with septic acute kidney injury. J Zhejiang Univ (Med Sci), 2018, 47(1): 41-50.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.02.06 或 http://www.zjujournals.com/med/CN/Y2018/V47/I1/41

表 1 实时定量RT-PCR引物序列

图 1 SAKI组与对照组血清炎症因子、肌酐和尿素氮表达水平比较

图 2 SAKI组与对照组肾脏组织病理学表现(HE染色)

图 3 SAKI组与对照组差异表达基因火山图

表 2 两组差异表达基因功能富集分析所得前20条相关信号通路

表 3 ATM信号通路相关差异表达基因

表 4 线粒体氧化应激和DNA损伤修复相关基因筛选结果

图 4 ATM信号通路相关差异表达基因与线粒体相关基因编码蛋白质的相互作用网络图

表 5 两组蛋白质相互作用相关基因RNA测序结果

图 5 SAKI组与对照组肾脏组织mtDNA损伤修复相关基因的表达水平比较

图 6 SAKI组与对照组肾脏组织中Gadd45α蛋白的表达

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