百草枯中毒小鼠肾脏损伤核心基因研究

Gene chip screen in mice kidney with acute paraquat poisoning and preliminary analysis of the differentially expressed genes

目的 应用基因芯片技术研究百草枯中毒小鼠模型肾脏损伤基因表达谱,通过基因信息学分析筛选核心基因,并以分子生物学方法给予验证.方法 C57BL/6雄性小鼠20只,随机（随机数字法）分成4组,分别为对照组（A组）和染毒组（B、C、D组）,每组5只.A组蒸馏水0.01 mL/g体质量灌胃,B、C、D组20％百草枯原液加蒸馏水稀释至0.05％,按0.01 mL/g体质量灌胃.A组于灌胃后即刻处理,B、C组分别于染毒6h、24h处理,D组观察48 h病死率,处理方法为麻醉后取出右肾组织样本,Affymetrix GeneChip Mouse Gene 1.0 ST Array芯片技术测定肾脏组织的基因表达谱,通过基因信息学分析筛选核心基因,并进行实时定量PCR和Western blot验证.结果 PQ灌胃后体质量明显下降,48 h病死率为100％.与对照组相比,PQ中毒6h组、24h组共有1 792个差异表达基因,基因信息学分析筛选出8个核心基因：Nlrc5、Serpinb9、Cd40、Rnf135、Dhx58、Sp110、Fcgr1、Arhgef12,进行PCR验证.Nlrc5、Rnf135、Serpinb9基因表达的差异有统计学意义（P＜0.05）.再对Nlrc5、Rnf135、Serpinb9进行Westem blot验证.均与对照组比较,中毒6h组,Nlrc5、Rnf135、Serpinb9表达上调差异有统计学意义（P＜0.05）,中毒24h组,Rnf135、Serpinb9表达上调差异有统计学意义（P＜0.05）,Nlrc5表达下调差异有统计学意义（P＜0.01）.不仅与基因芯片检测、信息学分析的结果一致,还证实了Nlrc5表达先上调（6 h）后下调（24 h）.结论 Nlrc5、Rnf135、Serpinb9是急性百草枯中毒小鼠肾脏损伤的核心基因,其参与肾脏损伤的具体机制有待进一步研究.

Objective To study the gene expression profile in mice kidney with acute paraquat （PQ） poisoning and identify key genes related to renal injury.Methods A total of 20 mice （C57BL/6） were randomly （random number） divided into four groups, namely control group （group A, n =5） and poisoned groups （groups B, C, D, n =5/group）.In group A, mice were administrated with distilled water （0.01 mL/g weight） while in groups B, C, D were administered with equivalent volume of PQ solution （diluted from 20% to 0.05% with distilled water） dissolved in distilled water via a gastric tube.Mice of group A were sacrificed immediately and mice of groups B and C at 6 h and 24 h after administration of PQ.The gene expression profile changes of kidney tissue were measured by cDNA Arraychip technology.Mice of group D were observed for mortality rate 48 h later.Results The body weights of mice decreased significantly after administration of PQ.The mortality in group D at 48 h after PQ poisoning was 100%.Compared with the control group, totally 1 792 genes with differential expression variations were identified in 6 h group and 24 h group.There were 8 key genes selected through bioinformatics analysis and they were arranged in real-time PCR： Nlrc5 , Serpinb9 , Cd40 , Rnf135 , Dhx58 , Spl 10 , Fcgrl , and Arhgef12.And then, Nlrc5 , Serpinb9 and Rnf135 were under Western blot investigation.The results of PCR and Western blot showed no significant difference to those from bioinformatics genetic analysis.Conclusions The investigation based on genome wide chip in researching related genes of PQ kidney has offered a novel idea in studying pathogenesis of acute PQ intoxication.