本文選題：阿霉素腎病 + 阿奇霉素　； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:通過(guò)觀察阿奇霉素(Azithromycin,AZM)對(duì)阿霉素腎病(Adriamycin-induced nephropathy,ADN)大鼠血、尿生化指標(biāo),腎組織基因表達(dá)譜的影響,從基因水平上探討阿霉素腎病的發(fā)病機(jī)制及阿奇霉素干預(yù)阿霉素腎病的機(jī)制。方法:135只健康雄性Wistar大鼠,適應(yīng)性喂養(yǎng)3d后,隨機(jī)分為空白組(A組)、模型組(B組)、阿奇霉素組(C組)、潑尼松組(D組)及聯(lián)合組(E組),每組27只。除空白組外,其余四組大鼠采用間隔1周2次尾靜脈注射阿霉素法(第1次注射4mg/kg,1周后第2次注射3.5mg/kg)建立阿霉素腎病大鼠模型,空白組同期注射同等劑量的生理鹽水。于實(shí)驗(yàn)第5周起,每日早晨給予阿奇霉素組、潑尼松組、聯(lián)合組相應(yīng)的干預(yù)藥物配成水溶液灌胃,其余兩組同期給予等劑量生理鹽水灌胃,各組大鼠均自由飲水及攝食基礎(chǔ)飼料。連續(xù)灌胃4周。測(cè)定各組大鼠第4周、6周、8周的24小時(shí)尿蛋白(24hUPro)排泄量、尿肌酐(Ucr)及血生化指標(biāo),包括血清總蛋白(Tp)、白蛋白(Alb)、膽固醇(Tcho)、血肌酐(Scr),計(jì)算內(nèi)生肌酐清除率(Ccr)。應(yīng)用SPSS22.0統(tǒng)計(jì)軟件對(duì)血、尿生化指標(biāo)的實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。利用Agilent公司生產(chǎn)的Agilent SurePrint G3 Rat Gene Expression(8×60k)基因芯片檢測(cè)第8周時(shí)各組大鼠的腎臟基因表達(dá)譜,并利用Gene Ontology富集分析和Pathway信號(hào)通路分析等生物信息學(xué)方法對(duì)檢測(cè)結(jié)果進(jìn)行分析。結(jié)果:1.五組大鼠24h尿蛋白定量及血生化結(jié)果實(shí)驗(yàn)第4周,B組、C組、D組和E組24hUPro100mg/d,提示造模成功,并且四組之間差異無(wú)統(tǒng)計(jì)學(xué)意義。1.1相同時(shí)間點(diǎn)A、B、C、D、E組之間血、尿生化指標(biāo)的比較實(shí)驗(yàn)第4周,B組、C組D組和E組較A組24hUPro、Scr、Tcho明顯升高(P0.05),Tp、Alb及Ccr明顯降低(P0.05)。實(shí)驗(yàn)第6周和第8周,C組、D組和E組24hUPro、Scr及Tcho明顯低于B組(P0.05),Tp、Alb及Ccr明顯高于B組(P0.05)。D組、E組24hUPro、Scr及Tcho低于c組(p0.05),tp、alb及ccr高于c組(p0.05)。e組24hupro、scr及tcho低于d組,tp、alb及ccr高于d組(p0.05)。1.2a、b、c、d、e組不同時(shí)間點(diǎn)血、尿生化指標(biāo)的比較a組4、6、8周各指標(biāo)之間差異無(wú)統(tǒng)計(jì)學(xué)意義。b組第6周24hupro、scr及tcho明顯高于第4周(p0.05),tp、alb及ccr明顯低于第4周(p0.05)。第8周24hupro、scr及tcho明顯高于第4周(p0.05)、第6周(p0.05),tp、alb及ccr明顯低于第4周(p0.05)、第6周(p0.05)。c、d、e組第6周24hupro、scr及tcho明顯低于各自第4周(p0.05),tp、alb及ccr明顯高于各自第4周(p0.05)。各組第8周24hupro、scr及tcho明顯低于第4周(p0.05)、第6周(p0.05),tp、alb及ccr明顯高于第4周(p0.05)、第6周(p0.05)。2.總rna質(zhì)檢結(jié)果利用qiagenrneasyminikit純化所有樣本總rna后,用nanodrop分光光度計(jì)及agilent2100bioanalyzer對(duì)樣本總rna的完整性及純度進(jìn)行定性和定量檢測(cè),經(jīng)檢測(cè)發(fā)現(xiàn),本實(shí)驗(yàn)所取樣本經(jīng)檢測(cè)均達(dá)到rin≥7.0,28s/18s0.7且a260/a280吸收率為1.9-2.2,提示總rna的純度及完整性達(dá)到實(shí)驗(yàn)要求,可以進(jìn)行芯片實(shí)驗(yàn)。3.差異基因篩選結(jié)果芯片雜交數(shù)據(jù)經(jīng)后期處理及分析發(fā)現(xiàn),與空白組相比,模型組共有185個(gè)基因發(fā)生差異性表達(dá),經(jīng)geneontology富集分析,上述差異表達(dá)的基因主要涉及的生物學(xué)過(guò)程有細(xì)胞周期、炎癥反應(yīng)及免疫反應(yīng)等,經(jīng)pathway信號(hào)通路分析,上述差異表達(dá)的基因涉及的信號(hào)通路有p53信號(hào)通路、b細(xì)胞受體信號(hào)通路、ampk信號(hào)通路、細(xì)胞因子-細(xì)胞因子受體通路等;而與模型組相比,阿奇霉素組共有824個(gè)基因發(fā)生差異性表達(dá),經(jīng)geneontology富集分析,上述差異表達(dá)的基因主要涉及的生物學(xué)過(guò)程包括上皮細(xì)胞的分化、脂肪酸代謝、損傷修復(fù)、吞噬作用的調(diào)節(jié)等,經(jīng)pathway信號(hào)通路分析,上述差異表達(dá)的基因涉及的信號(hào)通路有ppar-γ信號(hào)通路、camp信號(hào)通路、pi3k-akt信號(hào)通路等;與模型組相比,潑尼松組共有749個(gè)基因發(fā)生差異性表達(dá),經(jīng)geneontology富集分析,上述差異表達(dá)的基因主要涉及的生物學(xué)過(guò)程有淋巴細(xì)胞分化、炎癥反應(yīng)及免疫反應(yīng)等,經(jīng)pathway信號(hào)通路分析,上述差異表達(dá)的基因涉及的信號(hào)通路有細(xì)胞因子-細(xì)胞因子受體通路、ppar-γ信號(hào)通路、趨化因子信號(hào)轉(zhuǎn)導(dǎo)通路等;與模型組相比,聯(lián)合組共有537個(gè)基因發(fā)生差異性表達(dá),經(jīng)geneontology富集分析,上述差異表達(dá)的基因主要涉及的生物學(xué)過(guò)程包括細(xì)胞周期調(diào)控、免疫及防御反應(yīng)、損傷修復(fù)、對(duì)激素的反應(yīng)等,經(jīng)Pathway信號(hào)通路分析,上述差異表達(dá)的基因涉及的信號(hào)通路有PPAR-γ信號(hào)通路、細(xì)胞因子-細(xì)胞因子受體通路、PI3K-Akt信號(hào)通路等。結(jié)論:1.阿霉素腎病的發(fā)生涉及眾多基因的改變,上調(diào)表達(dá)的趨化因子CCL20、CXCL9、CXCL10、CXC3R及集落刺激因子CSF-1、腫瘤壞死因子超家族TNFSF9等參與的炎癥及免疫反應(yīng)在ADN發(fā)生中具有重要作用。p53信號(hào)通路可能通過(guò)促進(jìn)足細(xì)胞凋亡參與ADN的發(fā)生。2.阿奇霉素可能通過(guò)下調(diào)編碼炎癥介質(zhì)基因的表達(dá)控制炎癥反應(yīng),并在免疫損傷的修復(fù)中發(fā)揮一定作用。阿奇霉素通過(guò)影響PPAR-γ信號(hào)通路參與調(diào)節(jié)ADN大鼠脂質(zhì)的代謝。3.潑尼松干預(yù)可能會(huì)抑制TGF-β信號(hào)通路,延緩ADN大鼠腎臟纖維化的進(jìn)展。潑尼松干預(yù)可能通過(guò)上調(diào)VEGF基因的表達(dá)降低ADN大鼠尿蛋白水平。4.阿奇霉素與潑尼松兩者聯(lián)用能有效控制ADN大鼠炎癥反應(yīng),對(duì)損傷修復(fù)有一定作用,并能夠降低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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R726.9

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