顺铂诱导的小鼠肾间质纤维化模型中存在补体活化  

Complement activation in a mouse model of cisplatin-induced renal interstitial fibrosis

作　　者：冯敏[1,2] 曾红惠 廖伟棠 苏佳楠 Feng Min;Zeng Honghui;Liao Weitang;Su Jianan(Department of Nephrology,Sun Yat-sen Memorial Hospital of Sun Yat-sen University,Guangzhou 510120,China;Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation,Sun Yat-sen Memorial Hospital of Sun Yat-sen University,Guangzhou 510120,China)

机构地区：[1]中山大学孙逸仙纪念医院肾内科,广州510120 [2]中山大学孙逸仙纪念医院广东省恶性肿瘤表观遗传与基因调控重点实验室,广州510120

出　　处：《中华肾脏病杂志》2021年第10期809-816,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金(81800595)。

摘　　要：目的探索顺铂致肾间质纤维化的分子机制,为肾间质纤维化防治提供新思路。方法使用8周龄C57BL/6雄性无特定病原体级小鼠建模,将小鼠分为顺铂组和生理盐水组,每组6只,分别于第0、7、21天腹腔注射顺铂溶液(10 mg/kg)或生理盐水,第28天时处死小鼠,取肾组织行RNA Illumina高通量测序、实时定量PCR、Western印迹、Masson染色及生物信息学分析。结果通过实时定量PCR、Western印迹及Masson染色等验证,顺铂诱导的小鼠肾间质纤维化模型成功建立。RNA Illumina高通量测序结果显示,与生理盐水组相比,顺铂组差异表达长链非编码RNA(long noncoding RNA,lncRNA)有387个,通过实时定量PCR验证排名前两位差异表达lncRNA的表达,趋势均与测序结果一致;差异表达mRNA 2427个,其中补体C3在差异表达mRNA中排位第一。对测序结果行生物信息学分析,通过基因本体(GO)富集分析发现,与免疫相关的多个条目排位前20内;通过京都基因和基因组数据库(KEGG)富集分析发现,系统性红斑狼疮通路排位第一(ko05322,Q=3.4E-17),其中包含了补体级联反应通路。通过实时定量PCR验证发现,与生理盐水组比较,顺铂组补体C1q、C2、C3和C4 mRNA表达均升高(均P<0.05),与测序结果一致。结论小鼠周期性腹腔注射顺铂可诱导肾间质纤维化模型,该模型中肾脏补体通路活化。Objective To explore the mechanism of cisplatin-induced renal interstitial fibrosis and provide a new idea for the prevention and treatment of renal interstitial fibrosis.Methods Eight-week-old male C57BL/6 mice(specific pathogen-free)were used to carry out the experiment.The mice were divided into cisplatin group(10 mg/kg,n=6)and saline group(n=6)with intraperitoneal injection on day 0,7 and 21,and sacrificed on day 28.The kidney tissues were collected for RNA Illumina high-throughput sequencing,real-time PCR,Western blotting,Masson staining and bioinformatics analysis.Results Through real-time PCR,Western blotting and Masson staining,a mouse model with cisplatin-induced renal interstitial fibrosis was successfully established.Through RNA Illumina high-throughput sequencing,387 long noncoding RNA(lncRNA)and 2427 mRNA were differently expressed between cisplatin group and saline group.The expression of the top two lncRNA was confirmed by real-time PCR with the same tendency as RNA sequencing.Complement C3 was found to be at the top among the different expressed mRNA by RNA sequencing.Several terms related to immunity were found to be within the top 20 terms through Gene Ontology(GO)enrichment analysis.Systemic lupus erythematous pathway(ko05322,Q=3.4E-17),including the complement cascade pathway,was found to be the top pathway through Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The mRNA expression levels of C1q,C2,C3 and C4 were up-regulated remarkably in the cisplatin group by RNA sequencing than those in saline group(all P<0.05)and confirmed by real-time PCR.Conclusions Renal interstitial fibrosis can be induced by intraperitoneal injection of cisplatin periodically in mice,with complement cascade pathway activation in the diseased kidney.

关 键 词：顺铂 补体系统蛋白质类 计算生物学 肾间质纤维化 RNA高通量测序 

分 类 号：R692[医药卫生—泌尿科学]