阿奇霉素干预阿霉素肾病大鼠基因表达谱的研究

发布时间：2018-05-24 03:23

本文选题：阿霉素肾病 + 阿奇霉素　；参考：《天津医科大学》2016年硕士论文

【摘要】：目的:通过观察阿奇霉素(Azithromycin,AZM)对阿霉素肾病(Adriamycin-induced nephropathy,ADN)大鼠血、尿生化指标,肾组织基因表达谱的影响,从基因水平上探讨阿霉素肾病的发病机制及阿奇霉素干预阿霉素肾病的机制。方法:135只健康雄性Wistar大鼠,适应性喂养3d后,随机分为空白组(A组)、模型组(B组)、阿奇霉素组(C组)、泼尼松组(D组)及联合组(E组),每组27只。除空白组外,其余四组大鼠采用间隔1周2次尾静脉注射阿霉素法(第1次注射4mg/kg,1周后第2次注射3.5mg/kg)建立阿霉素肾病大鼠模型,空白组同期注射同等剂量的生理盐水。于实验第5周起,每日早晨给予阿奇霉素组、泼尼松组、联合组相应的干预药物配成水溶液灌胃,其余两组同期给予等剂量生理盐水灌胃,各组大鼠均自由饮水及摄食基础饲料。连续灌胃4周。测定各组大鼠第4周、6周、8周的24小时尿蛋白(24hUPro)排泄量、尿肌酐(Ucr)及血生化指标,包括血清总蛋白(Tp)、白蛋白(Alb)、胆固醇(Tcho)、血肌酐(Scr),计算内生肌酐清除率(Ccr)。应用SPSS22.0统计软件对血、尿生化指标的实验数据进行统计学分析。利用Agilent公司生产的Agilent SurePrint G3 Rat Gene Expression(8×60k)基因芯片检测第8周时各组大鼠的肾脏基因表达谱,并利用Gene Ontology富集分析和Pathway信号通路分析等生物信息学方法对检测结果进行分析。结果:1.五组大鼠24h尿蛋白定量及血生化结果实验第4周,B组、C组、D组和E组24hUPro100mg/d,提示造模成功,并且四组之间差异无统计学意义。1.1相同时间点A、B、C、D、E组之间血、尿生化指标的比较实验第4周,B组、C组D组和E组较A组24hUPro、Scr、Tcho明显升高(P0.05),Tp、Alb及Ccr明显降低(P0.05)。实验第6周和第8周,C组、D组和E组24hUPro、Scr及Tcho明显低于B组(P0.05),Tp、Alb及Ccr明显高于B组(P0.05)。D组、E组24hUPro、Scr及Tcho低于c组(p0.05),tp、alb及ccr高于c组(p0.05)。e组24hupro、scr及tcho低于d组,tp、alb及ccr高于d组(p0.05)。1.2a、b、c、d、e组不同时间点血、尿生化指标的比较a组4、6、8周各指标之间差异无统计学意义。b组第6周24hupro、scr及tcho明显高于第4周(p0.05),tp、alb及ccr明显低于第4周(p0.05)。第8周24hupro、scr及tcho明显高于第4周(p0.05)、第6周(p0.05),tp、alb及ccr明显低于第4周(p0.05)、第6周(p0.05)。c、d、e组第6周24hupro、scr及tcho明显低于各自第4周(p0.05),tp、alb及ccr明显高于各自第4周(p0.05)。各组第8周24hupro、scr及tcho明显低于第4周(p0.05)、第6周(p0.05),tp、alb及ccr明显高于第4周(p0.05)、第6周(p0.05)。2.总rna质检结果利用qiagenrneasyminikit纯化所有样本总rna后,用nanodrop分光光度计及agilent2100bioanalyzer对样本总rna的完整性及纯度进行定性和定量检测,经检测发现,本实验所取样本经检测均达到rin≥7.0,28s/18s0.7且a260/a280吸收率为1.9-2.2,提示总rna的纯度及完整性达到实验要求,可以进行芯片实验。3.差异基因筛选结果芯片杂交数据经后期处理及分析发现,与空白组相比,模型组共有185个基因发生差异性表达,经geneontology富集分析,上述差异表达的基因主要涉及的生物学过程有细胞周期、炎症反应及免疫反应等,经pathway信号通路分析,上述差异表达的基因涉及的信号通路有p53信号通路、b细胞受体信号通路、ampk信号通路、细胞因子-细胞因子受体通路等;而与模型组相比,阿奇霉素组共有824个基因发生差异性表达,经geneontology富集分析,上述差异表达的基因主要涉及的生物学过程包括上皮细胞的分化、脂肪酸代谢、损伤修复、吞噬作用的调节等,经pathway信号通路分析,上述差异表达的基因涉及的信号通路有ppar-γ信号通路、camp信号通路、pi3k-akt信号通路等;与模型组相比,泼尼松组共有749个基因发生差异性表达,经geneontology富集分析,上述差异表达的基因主要涉及的生物学过程有淋巴细胞分化、炎症反应及免疫反应等,经pathway信号通路分析,上述差异表达的基因涉及的信号通路有细胞因子-细胞因子受体通路、ppar-γ信号通路、趋化因子信号转导通路等;与模型组相比,联合组共有537个基因发生差异性表达,经geneontology富集分析,上述差异表达的基因主要涉及的生物学过程包括细胞周期调控、免疫及防御反应、损伤修复、对激素的反应等,经Pathway信号通路分析,上述差异表达的基因涉及的信号通路有PPAR-γ信号通路、细胞因子-细胞因子受体通路、PI3K-Akt信号通路等。结论:1.阿霉素肾病的发生涉及众多基因的改变,上调表达的趋化因子CCL20、CXCL9、CXCL10、CXC3R及集落刺激因子CSF-1、肿瘤坏死因子超家族TNFSF9等参与的炎症及免疫反应在ADN发生中具有重要作用。p53信号通路可能通过促进足细胞凋亡参与ADN的发生。2.阿奇霉素可能通过下调编码炎症介质基因的表达控制炎症反应,并在免疫损伤的修复中发挥一定作用。阿奇霉素通过影响PPAR-γ信号通路参与调节ADN大鼠脂质的代谢。3.泼尼松干预可能会抑制TGF-β信号通路,延缓ADN大鼠肾脏纤维化的进展。泼尼松干预可能通过上调VEGF基因的表达降低ADN大鼠尿蛋白水平。4.阿奇霉素与泼尼松两者联用能有效控制ADN大鼠炎症反应,对损伤修复有一定作用,并能够降低ADN大鼠的蛋白尿水平。
[Abstract]:Objective: To investigate the effect of Azithromycin (AZM) on the blood, urine biochemical indexes and renal tissue gene expression profiles of adriamycin (Adriamycin-induced nephropathy, ADN) rats. The mechanism of adriamycin nephrosis and the mechanism of azithromycin induced adriamycin nephropathy were investigated from the gene level. Methods: 135 healthy males were Wistar large. Rats, after adaptive feeding 3D, were randomly divided into blank group (group A), model group (group B), azithromycin group (group C), prednisone group (group D) and group E (group E), with 27 rats in each group. The other four groups of rats were treated with adriamycin (first injections of 4mg/kg, second times 3.5mg/kg after 1 weeks after 1 weeks) to establish adriamycin nephrotic rats. After fifth weeks, the group of azithromycin group, prednisone group, combined group of intervention drugs were fed with water solution, and the other two groups were given the same dosage of physiological saline for the same period. The rats in each group were free drinking water and feeding basic feed for 4 weeks. The excretion of urine protein (24hUPro), urinary creatinine (Ucr) and blood biochemical indexes, including serum total protein (Tp), albumin (Alb), cholesterol (Tcho), serum creatinine (Scr), and endogenous creatinine clearance (Ccr) were calculated for fourth weeks, 6 weeks and 8 weeks. The statistical analysis of experimental data on blood and urine biochemical indexes should be used in SPSS22.0 statistical software. Ag The Agilent SurePrint G3 Rat Gene Expression (8 x 60K) gene chip produced by ilent company was used to detect the renal gene expression profiles of rats in each group at eighth weeks. The results were analyzed by the bioinformatics methods such as Gene Ontology enrichment analysis and Pathway signal pathway analysis. Results: the quantitative and blood biochemistry of 24h proteinuria in 1. five groups of rats Results fourth weeks, B group, C group, D group and E group 24hUPro100mg/d, suggesting that the model was successful, and there was no statistically significant difference between the four groups in the same time point A, B, C, D, E group of blood and urine biochemical indexes for fourth weeks. Sixth weeks and eighth weeks, 24hUPro, Scr and Tcho in group C, D group and E group were significantly lower than B group (P0.05), Tp, Alb and Ccr were higher than those of the B group. There was no significant difference between the a group and the 4,6,8 weeks of the a group, and the SCR and TCHO were significantly higher than that of fourth weeks (P0.05), TP, ALB and CCR were obviously lower than fourth weeks (P0.05). Eighth weeks 24hupro was significantly higher than that of fourth weeks. Sixth weeks were obviously lower than fourth weeks, sixth weeks. SCR and TCHO were significantly lower than their fourth weeks (P0.05), TP, ALB and CCR were significantly higher than their respective fourth weeks (P0.05). The eighth weeks 24hupro, SCR and TCHO were significantly lower than fourth weeks (P0.05), sixth weeks (P0.05). P spectrophotometer and agilent2100bioanalyzer test the integrity and purity of sample total RNA qualitatively and quantitatively. It is found that the samples obtained from this experiment have reached Rin more than 7.0,28s/18s0.7 and a260/a280 absorption rate is 1.9-2.2, suggesting that the purity and integrity of the total RNA have reached the experimental requirements, and the.3. difference of the chip experiment can be carried out. After the late processing and analysis of the hybridization data of the gene screening result chip, it was found that compared with the blank group, there were 185 differentially expressed genes in the model group, and enriched by geneontology. The main biological processes mentioned above were cell cycle, inflammatory reaction and immune response, and were analyzed by pathway signaling pathway. The signaling pathways involved in differentially expressed genes include p53 signaling pathway, B cell receptor signaling pathway, AMPK signaling pathway, cytokine receptor pathway and so on. Compared with the model group, there are 824 differentially expressed genes in the azithromycin group, and the genes expressed above are mainly involved in the genetic diversity analysis. The physical process includes epithelial cell differentiation, fatty acid metabolism, injury repair and regulation of phagocytosis. Through pathway signal pathway analysis, the signal pathways involved in these differentially expressed genes include ppar- gamma signaling pathway, cAMP signaling pathway, PI3K-Akt signaling pathway, and so on. Compared with the model group, the prednisone group has 749 genes. The above expressed genes mainly involve lymphocyte differentiation, inflammatory reaction and immune response, and are analyzed by pathway signaling pathway. The signal pathways involved in these differentially expressed genes include cytokine cytokine receptor pathway, ppar- gamma signaling pathway, chemokine signaling pathway, and the expression of genes involved in geneontology analysis. Compared with the model group, there were 537 differentially expressed genes in the combined group and enriched by geneontology. The above expressed genes mainly involved biological processes including cell cycle regulation, immune and defense response, injury repair, response to hormone, and Pathway signaling pathway analysis. The signal pathways involved are PPAR- gamma signaling pathway, cytokine receptor pathway, PI3K-Akt signaling pathway, etc. conclusion: 1. adriamycin nephrosis involves a variety of gene changes, up-regulated chemokine CCL20, CXCL9, CXCL10, CXC3R and colony stimulating factor CSF-1, tumor necrosis factor superfamily TNFSF9 and so on. Inflammatory and immune responses play an important role in the development of ADN..p53 signaling pathway may be involved in the occurrence of ADN by promoting the apoptosis of podocyte.2., and azithromycin may control the inflammatory response by down regulation of the expression of the genes encoding the inflammatory mediators and play a role in the repair of immune injury. Azithromycin affects PPAR- gamma signaling. Pathway involved in regulating lipid metabolism in ADN rats.3. prednisone intervention may inhibit the TGF- beta signaling pathway and delay the progression of renal fibrosis in ADN rats. The prednisone intervention may reduce the protein level of ADN rats by up regulation of the VEGF gene, and the combination of.4. azithromycin and prednisone can effectively control the inflammatory response of ADN rats, and the damage to the injury of ADN rats can be effectively controlled. Repair has a certain effect and can reduce the level of proteinuria in ADN rats.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R726.9

【参考文献】