摘要
目的寻找脓毒症相关性急性肾损伤(AKI)的关键基因, 为脓毒症相关性AKI的治疗提供理论和实验依据。方法①生物信息学分析:对基因表达数据库(GEO)中GSE30718和GSE53773数据集进行生物信息学分析, 这两个数据集记录了肾移植手术前后对移植肾进行规律性肾活检的基因芯片数据, 一部分患者在肾移植后发生了AKI。对两个数据集中AKI相关基因表达差异进行分析, 找到在两个数据集中差异一致且受试者工作特征曲线下面积(AUC)较高的基因作为目的基因, 进行后续细胞验证实验。②细胞验证实验:体外培养人近端肾小管上皮细胞株HK2, 使用10 mg/L脂多糖(LPS)孵育6 h制备LPS-HK2细胞模型(LPS模型组), 并设空白对照组;使用小干扰RNA(siRNA)技术对LPS-HK2细胞中通过生物信息学分析得出的目的基因进行敲减(基因敲减组), 并设置基因阴性对照组。采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测HK2细胞中目的基因的表达;采用酶联免疫吸附试验(ELISA)检测细胞培养液中炎症因子水平;采用蛋白质免疫印迹试验(Western blotting)检测细胞中凋亡关键蛋白的表达。结果①生物信息学分析结果:两个数据集间有325个基因呈现相同的表达趋势, 其中144个显著下调, 181个显著上调, 而分泌性白细胞蛋白酶抑制因子(SLPI)在两个数据集中表达差异的统计学意义均较大;进一步受试者工作特征曲线(ROC曲线)分析证实, GSE30718和GSE53773数据集中SLPI表达量对AKI的诊断效能均较大, AUC分别为0.83和0.92。故选择SLPI作为目的基因进行后续细胞验证实验。②细胞验证实验结果:RT-qPCR结果显示, LPS模型组细胞中SLPI表达较空白对照组显著升高(2^(-ΔΔCT):1.80±0.14比1.00±0.11, P<0.01), 变化趋势与生物信息学分析结果相符。进一步敲减SLPI基因分析显示, 基因敲减组LPS-HK2细胞培养液中炎症因子水平较阴性对照组显著上调〔白细胞介素-6(IL-6, ng/L):509.58±27.08比253.87±75.83, IL-1β(ng/L):490.99±49.52比239.67±26.97, 肿瘤坏死因子-α(TNF-α, ng/L):755.22±48.66比502.06±10.92, 均P<0.01〕, 说明SLPI可抑制LPS诱导的HK2细胞炎症反应;基因敲减组LPS-HK2细胞凋亡关键蛋白Bax、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)表达均较阴性对照组显著升高〔Bax蛋白(Bax/GAPDH):1.38±0.12比1.00±0.10, caspase-3蛋白(caspase-3/GAPDH):1.44±0.15比1.00±0.11, 均P<0.05〕, 而Bcl-2表达显著降低(Bcl-2/GAPDH:0.83±0.08比1.00±0.05, P<0.05), 说明SLPI可抑制细胞在炎症反应中的凋亡。结论 SLPI能抑制LPS诱导的HK2细胞炎症反应和凋亡, 可能参与肾小管细胞在脓毒症反应中的保护机制, 成为脓毒症相关性AKI治疗的潜在靶点。
Objective To screen out the potential key genes of sepsis-associated acute kidney injury(AKI),and provide theoretical and experimental evidence for the treatment of sepsis-associated AKI.Methods①Bioinformatics analysis:two gene expression datasets(GSE30718 and GSE53773)were downloaded for bioinformatics analysis from the Gene Expression Omnibus(GEO).These two datasets recorded mRNA microarray data from kidney biopsies before and after kidney transplantation,and a subset of patients developed AKI after kidney transplantation.Differential analysis was conducted,and the genes with the same differential expression and a higher area under the receiver operator characteristic curve(AUC)in both databases were used as the target gene for subsequent cell experiments.②Cell validation experiment:human proximal renal tubular cells HK2 were cultured in vitro,and lipopolysaccharide(LPS)was used for establishing LPS-HK2 cell model(LPS 10 mg/L for 6 hours,LPS model group),and the blank control group was set.Then,small interfering RNA(siRNA)technology was used to knock down the target gene obtained by bioinformatics analysis in LPS-HK2 cells(gene knockdown group),and a gene negative control group was set.The real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-qPCR)technique was used to detect the expression of the target gene in HK2 cells.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors in the cell supernatants.Western blotting was used to detect the expressions of key apoptosis proteins.Results①Results of bioinformatics analysis:325 genes in the two datasets showed the same expression trend,of which 144 were significantly down-regulated and 181 were significantly up-regulated,while the expression difference of secretory leukocyte protease inhibitor(SLPI)in the two datasets was both statistically significant.Further receiver operator characteristic curve(ROC curve)analysis confirmed that the SLPI expression in GSE30718 and GSE53773 datasets had a high diagnostic efficiency for AKI,with AUC of 0.83 and 0.92,respectively.Therefore,SLPI was selected as the target gene for subsequent cell validation experiment.②Cell validation experiment:the RT-qPCR analysis showed that the expression of SLPI in LPS-HK2 cells of the LPS model group was significantly higher than that of the blank control group(2^(-ΔΔCT):1.80±0.14 vs.1.00±0.11,P<0.01),and the change trend was the same with the results of bioinformatics analysis.Furthermore,knockdown SLPI gene analysis showed that the levels of inflammatory factors in LPS-HK2 cells supernatants in the gene knockdown group were significantly higher than those in the negative control group[Interleukin-6(IL-6,ng/L):509.58±27.08 vs.253.87±75.83,IL-1β(ng/L):490.99±49.52 vs.239.67±26.97,tumor necrosis factor-α(TNF-α,ng/L):755.22±48.66 vs.502.06±10.92,all P<0.01].The above results indicated that SLPI could inhibit the inflammatory response of HK2 cells induced by LPS.The expressions of key apoptosis proteins Bax and caspase-3 in LPS-HK2 cells in the gene knockdown group were significantly higher than those in the negative control group[Bax protein(Bax/GAPDH):1.38±0.12 vs.1.00±0.10,caspase-3 protein(caspase-3/GAPDH):1.44±0.15 vs.1.00±0.11,both P<0.05],and Bcl-2 expression was significantly decreased(Bcl-2/GAPDH:0.83±0.08 vs.1.00±0.05,P<0.05),the above results indicated that SLPI could inhibit the apoptosis of cells in the inflammatory response.Conclusion SLPI can inhibit the inflammatory response and apoptosis of HK2 cells induced by LPS,which may be involved in the protective mechanism of renal tubular cells in the response to sepsis,and is a potential target for the treatment of sepsis-associated AKI.
作者
张德龙
汪文杰
邵仁德
汪洋
吴忠展
鲁厚清
Zhang Delong;Wang Wenjie;Shao Rende;Wang Yang;Wu Zhongzhan;Lu Houqing(Department of Intensive Care Unit,Tongling People's Hospital,Tongling 244000,Anhui,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2022年第7期721-726,共6页
Chinese Critical Care Medicine
基金
安徽省卫生健康委科研项目(AHWJ2021b092)
安徽省铜陵市科技计划项目(20200203041)。