【作者】 吴迪；

【导师】 姜艳芳；

【作者基本信息】 吉林大学 ， 免疫学， 2020， 博士

【摘要】 背景与目的:膜性肾病（membranous nephropathy,MN）是常见的自身免疫性肾小球肾炎,以上皮下的免疫复合物沉积为主要特征。近年来,MN在我国等发展中国家的发病率呈逐年上升趋势。尽管有三分之一左右的MN患者可自发缓解,但仍有约40%的患者可在十年内进展为终末期肾病（end-stage renal disease,ESRD）,严重影响患者的生存质量。目前MN的诊断主要依靠有创的肾组织活检技术,用于MN治疗的药物以类固醇、烷化剂、钙调神经磷酸酶抑制剂及利妥昔单抗为主,尚无特异的治疗方法。因此,寻求有效的诊断标志物及特异性治疗靶标是MN研究中亟需解决的问题。基因组学分析被视为系统生物学研究的里程碑,是鉴定生物标志物和阐明疾病病理机制的有效方法。其中芯片检测及分析已经应用于研究人类的多种肾脏疾病,如肾细胞癌、糖尿病肾病、急性肾损伤和肾移植排斥等。Hauser等人对实验性膜性肾病基因芯片的研究揭示了肾脏细胞的DNA损伤和修复,以及细胞外间质的变化是疾病的主要病理过程。然而,MN的发病机制及相关的分子生物学原因仍不完全明确。本文应用人类全基因组表达谱芯片技术对MN患者与健康人肾皮质的基因进行差异表达分析和富集分析,拟探索在MN中异常表达的基因及其相关的生物学过程和代谢通路;并进一步探索显著影响这些过程或通路的核心基因,以期了解MN的潜在分子机制,为MN的诊疗提供新的思路。方法:1.研究对象:本研究共纳入14例原发性膜性肾病（idiopathic MN,i MN）患者,其中包含i MNⅠ-Ⅱ期患者（MNⅠ-Ⅱ组）7人,i MNⅢ期患者（MNⅢ组）7人。所有患者均经肾穿刺病理活检确诊为i MN,并根据组织形态学及免疫荧光表达分为MNⅠ-Ⅱ及MNⅢ两组。肾皮质活检组织均取自患者初次治疗之前。另有正常对照者（HC组）7人。2.全基因组表达谱芯片杂交:每组收集3例肾皮质活检组织,应用Mini BEST Universal RNA Extraction试剂盒分别提取各样本的总RNA。应用Illumina全基因组表达谱芯片分别对各样本的总RNA进行芯片杂交实验。分析MNⅠ-Ⅱ组vs HC组及MNⅢvs HC组的差异表达基因（differentially expressed genes,DEGs）,对MNⅠ-Ⅱ组vs HC组的DEGs和MNⅢ组vs HC组的DEGs取交集,以获得在两个比较组中均差异表达的基因,即共同差异表达基因（overlapping differentially expressed genes,ODEGs）。3.生物信息学分析:利用GO（Gene Ontology）数据库对ODEGs进行生物学功能（Biological process,BP）富集分析,利用KEGG（Kyoto Encyclopedia of Genes and Genomes）数据库对ODEGs行通路富集分析,进一步筛选核心基因,并对全部核心基因进行文献检索分析。4.血脂浓度测定:采集MNⅠ-Ⅱ组、MNⅢ组患者及HC组空腹静脉血,应用GPO-PAP法检测血清甘油三酯（TG）水平,应用CHOD-PAP法检测血清总胆固醇（TCHOL）浓度,应用直接法-过氧化氢酶清除法检测血清高密度脂蛋白胆固醇（HDLc）及低密度脂蛋白胆固醇（LDLc）的浓度。进一步分析血清TG、TCHOL、HDLc及LDLc与核心基因的m RNA信号强度的相关性。5.RT-PCR验证:为进一步验证表达谱芯片数据,收集MNⅠ-Ⅱ组、MNⅢ组及HC组各4例肾皮质活检组织,利用RT-PCR测定PLA2G12B在各样本的m RNA水平,以ACTB为内参,采用2^-ΔΔCt法计算目的基因的相对表达水平。6.免疫荧光染色:为验证关键基因PLA2G12B编码蛋白在MN患者肾皮质中表达的变化,对MNⅠ-Ⅱ组、MNⅢ组及HC组的全部肾皮质活检石蜡标本进行切片,利用免疫荧光染色评估PLA2G12B蛋白在各组肾皮质中的表达量。7.PLA2G12B蛋白体外刺激足细胞:为探讨PLA2G12B蛋白对人足细胞的作用,体外培养人足细胞,以PLA2G12B蛋白按照时间梯度予以刺激,分为对照组（0h）、0.5h、1h、2h及6h组,分别以流式细胞仪检测足细胞的凋亡情况,以高效液相色谱-质谱法检测培养基中的花生四烯酸水平。8.统计学分析:应用SPSS 21.0软件进行统计学分析。组间差异分析应用Welch’s t检验。相关性分析应用Pearson秩相关性检验。p值<0.05被认为具有统计学意义。结果:1.MNⅠ-Ⅱ组与HC组比较,有291个上调基因及659个下调基因;MNⅢ组与HC组比较,上调及下调的基因数分别为217及382个。在MNⅠ-Ⅱ组vs HC组与MNⅢ组vs HC组的共同差异表达基因中,上调者为167个,下调者为291个。2.通过GO BP富集分析,发现上调的ODEGs主要富集在甘油三酯代谢、丙氨酸分解代谢及乙醛酸分解代谢等代谢相关过程,而下调的ODEGs主要富集在白细胞活化、白细胞细胞间粘附及白细胞趋化作用等炎症反应相关过程。3.进一步对ODEGs行KEGG通路富集分析,结果显示上调的ODEGs参与16个KEGG通路,包括胆固醇代谢、脂肪的消化及吸收、花生四烯酸代谢等。下调的ODEGs参与13个信号通路,包括细胞-细胞因子受体相互作用、IL-17信号通路及NF-κB信号通路等。4.从上述GO BP及KEGG通路富集项中共筛选出38个核心基因,包括PLA2G12B（phospholipase A2 groupⅫ）、APOA1（apolipoprotein A1）、APOB（apolipoprotein B）、APOC3（apolipoprotein C3）及CETP（cholesteryl ester transfer protein）等与脂质代谢相关的基因。5.MN患者肾皮质中的PLA2G12B、CD8A（CD8a molecule）、LTB（lymphotoxin beta）及NLRP3（NLR family pyrin domain containing 3）等m RNA信号强度与患者血脂参数相关。6.RT-PCR检测显示PLA2G12B的m RNA水平在MN患者肾皮质中较正常对照组增加,与芯片结果一致。7.免疫荧光检测结果显示在MNⅠ-Ⅱ组与MNⅢ组中PLA2G12B的表达均较在HC组中显著升高,而在MNⅠ-Ⅱ组与MNⅢ组间并无统计学差异。8.PLA2G12B体外刺激足细胞,发现0.5h组细胞凋亡与对照组无显著差异,1h、2h及6h组细胞凋亡的百分比均较对照组有显著增加,且随作用时间的增加,凋亡细胞百分比呈递增趋势。9.花生四烯酸浓度检测结果显示,0.5h组及1h组的花生四烯酸浓度与对照组之间无显著差异,而2h组及6h组的浓度均较对照组有显著升高。结论:1.脂质代谢相关代谢通路,包括胆固醇代谢及花生四烯酸代谢等在MN的致病机制中具有重要作用。2.免疫反应相关代谢通路,包括IL-17信号通路及NF-κB信号通路等可能是MN致病机制中的重要代谢通路。3.PLA2G12B可在体外诱导人足细胞凋亡。4.PLA2G12B可能通过花生四烯酸代谢导致MN中足细胞的损伤。更多还原

【Abstract】 Background and objective:Membranous nephropathy（MN）is a common autoimmune glomerulonephritis characterized by the deposition of immune complexes in the subepithelial space.Although one-third of MN patients achieve spontaneous remission,approximately 40%patients progress to end-stage renal disease（ESRD）in ten years,seriously affecting the quality of life of patients.At present,the diagnosis of MN mainly depends on invasive renal biopsy technique,so it is urgent to seek effective diagnostic markers and specific therapeutic targets on MN.Genomic analyses,which represent major cornerstones of systems biology research,are considered to be unbiased methods for identifying biomarkers and elucidating pathological mechanisms of disease.For example,microarray analysis has been applied to investigate several kidney diseases in humans,such as renal cell carcinoma,diabetic kidney disease,acute kidney injury,and renal graft rejection.In the field of MN,Hauser et.al reported microarray analysis of gene expression in experimental membranous nephropathy and revealed that DNA damage and repair,as well as changes in the extracellular matrix,are major processes in the disease.Nevertheless,the exact pathogenic mechanisms and molecular events underlying MN development remain poorly understood.Thus,the aim of this study was to identify differential gene expression profiles of the MN renal cortex compared to healthy humans and to identify the underlying molecular basis of MN.We performed a whole-genome DNA microarray assay and enrichment analysis to identify relevant biological processes and metabolic pathways regulated by abnormally expressed genes;we also investigated hub genes that may significantly impact those processes or pathways.The data presented herein enhance our knowledge of MN at the genetic level,provide insight into MN pathogenesis,and implicate a new therapeutic strategy for slowing disease progression.Methods:1.Study population.A total of 14 idopathic MN cases were recruited in this study,including seven i MN I–II（Group MN I–II）and seven i MN III（Group MN III）.All the patients were diagnosed and ranked grade I–II or III according to histopathological morphology and immunofluorescence.Punctures of the renal cortex were sampled from each patient before any treatment.Seven healthy controls（Group HC）were recruited.2.Whole-Genome Gene Expression Profiling microarray hybridization.Three puncture samples of the renal cortex from each group were recruited.Total RNA was extracted from each samples using Mini BEST Universal RNA Extraction Kit.Microarray hybridization were performed with total RNA using Illuminar Whole-Genome Gene Expression Directed Hybridization System.Differentially expressed genes（DEGs）between MN I–II vs HC and MN III vs HC were identified.Overlapping differentially expressed genes（ODEGs）were identified in the genes with altered expression in both comparisons.3.Using the Gene Ontology（GO）data to perform BP enrichment analyses and filting hub genes according to the correlation.Using the Kyoto Encyclopedia of Genes and Genomes（KEGG）data to perform pathway enrichment analyses and filting hub genes that shared between KEGG terms.All the hub genes were analyzed by literature search.4.Determination of blood lipid concentration.The fasting venous blood samples were collected from patients in group MN I-II,group MN III and group HC,the level of serum triglyceride（TG）was detected by GPO-PAP method,and the level of serum total cholesterol（TCHOL）was detected by CHOD-PAP method,serum high-density lipoprotein cholesterol（HDLc）and low-density lipoprotein cholesterol（LDLc）were measured by direct method（catalase clearance method）.Using Prism software to analyze the correlation of m RNA levels of hub genes in the renal cortex with circulating TG、TCHOL、HDLc and LDLc concentrations.5.RT-PCR were performed with four total RNA samples in each group using One Step SYBR?Prime Script?RT-PCR Kit II.ACTB was used as internal parameter and 2^-ΔΔCt method was used to calculate the relative expression of PLA2G12B.6.Immunofluorescence staining:All renal cortex biopsies of patients with MNⅠ-Ⅱ,MN III and HC were sectioned,and expression of PLA2G12B protein in MN and HC renal cortex was evaluated by immunofluorescence staining.7.PLA2G12B protein stimulates podocytes in vitro.In order to investigate the effect of PLA2G12B protein on podocytes,human podocytes were cultured in vitro and stimulated with PLA2G12B protein according to time gradient.They were divided into control group（0 h）,0.5 h,1 h,2 h and 6 h groups.The apoptosis of podocytes was detected by flow cytometry,detection of arachidonic acid levels in culture media by high-performance liquid chromatography-mass spectrometry.8.Statistical Analysis.Using SPSS 21.0 software for statistical analysis.Welch’s t test was used for inter-group difference analysis.Pearson rank correlation test was used for correlation analysis.P<0.05was considered statistically significant.Results:1.The MN I-II vs.HC comparison detected 291 up-and 659down-regulated genes,meanwhile,in the MN III vs.HC comparison,217 up-and 382 down-regulated genes were detected.Furthermore,167 up-and 291down-regulated genes overlapped between the dysregulated genes in both comparisons.2.Upregulated GO BP enrichment terms,such as triglyceride metabolic process,alanine catabolic process and glyoxylate catabolic process,are related to metabolic processes,whereas the downregulated terms,such as leukocyte activation,leukocyte cell-cell adhesion and leukocyte chemotaxis,are mainly associated with immunologic processes.3.Upregulated ODEGs participate in 16 KEGG pathway,such as cholesterol metabolism,fat digestion and absorption,and the PPAR signaling pathway.Conversely,downregulated ODEGs participate in 13pathways,such as cytokine-cytokine receptor interaction,the IL-17signaling pathway and NF-kappa B signaling pathway.4.38 hub genes were filtered from GO BP and KEGG pathway terms,including the genes PLA2G12B（phospholipase A2 groupⅫ）,APOA1（apolipoprotein A1）,APOB（apolipoprotein B）,APOC3（apolipoprotein C3）and CETP（cholesteryl ester transfer protein）,which associated with lipid metabolism.5.Several m RNA signal intensities,such as PLA2G12B,CD8A（CD8a molecule）,LTB（lymphotoxin beta）,and NLRP3（NLR family pyrin domain containing 3）were associated with dyslipidemia.6.RT-PCR showed an elevated m RNA levels of PLA2G12B among the MN patients,which were consistent with the microarray result.7.The results of immunofluorescence assay showed that the expression of PLA2G12B was significantly higher in MN I-II group and MN III group than in HC group,but there was no significant difference between MN I-II group and MN III group.8.PLA2G12B stimulated podocytes in vitro.There was no significant difference in apoptosis between 0.5 h group and control group.The percentage of apoptosis in 1h,2h and 6h groups was significantly higher than that in control group,the percentage of apoptotic cells increased gradually.9.The arachidonic acid detection results showed 0.5 h group and 1h group had no significant difference with the control group,but the concentration of arachidonic acid in 2h group and 6h group was significantly higher than the control group.Conclusions:1.The pathways and biological processes related to lipid metabolism,such as cholesterol metabolism and arachidonic acid metabolism play an important role in MN.2.The metabolic pathways related to immune activation,such as IL-17and NF-kappa B signaling pathways are involved in the pathological process of MN.3.PLA2G12B can induce podocyte apoptosis in vitro.4.The podocyte injury in MN maybe induced by PLA2G12B through arachidonic acid metabolism.更多还原