【作者】 林华；

【导师】 赵丰丽； 眭维国； 戴勇；

【作者基本信息】 广西师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 系统性红斑狼疮(Systemic lupus erythematosus, SLE)是一种累及全身多个系统的难以预料的自身免疫性疾病,其最常见和最严重的并发症为狼疮性肾炎(Lupus nephritis),临床统计SLE患者中有肾损害的达到95%,而病理活检结果更是接近100%。自从糖皮质激素与免疫抑制剂被用于治疗SLE后,患者的预后虽已经有明显改善,但总的来说该病治疗现状仍不容乐观,有约15%的患者在发病5年内死亡和出现肾功能衰竭,同时复发也是治疗过程中面临着的一个严峻问题。而肾移植是目前终末期肾病患者最理想、最经济的一种治疗方法,但移植后急性排斥反应严重影响着移植物及受者的存活。目前,移植肾活检是能明确诊断排斥反应的唯一方法,但此法即昂贵又不方便还易引起肾损伤导致各种并发症。因此新的有效、方便的SLE和排斥反应早期生物标记的发现,将能大大提高SLE与肾移植治疗效率。在此方面研究人员做了大量工作,当前临床上治疗SLE普遍采用糖皮质激素和／或免疫抑制剂从整体抑制机体的免疫功能,对该病存有的免疫功能紊乱可谓是没有目标性,缺乏靶向性。同时这些药物副作用大,很多患者由于副作用而被迫停药,致使治疗中断。在排斥反应方面,主要方向为使用先进的科学技术从蛋白尿和患者外周血中寻找排斥反应的早期生物标记,但目前这些研究成果都还未能运用于临床上转录因子(Transcription factor,TF)又称为反式作用因子,是能够与真核基因启动子区域中顺式作用元件发生特异性相互作用的DNA结合蛋白,通过它们之间以及与其它相关蛋白之间的相互作用,激活或抑制转录。一个转录因子可调控数百个基因的表达,而一个基因的表达又可受数个转录因子的调控。转录因子的结构和功能缺陷,与人类的一些疾病(如肿瘤和炎症)相关,许多转录因子还是药物作用的靶点。新的生物标记能及时、可靠、特异地预示SLE患者和移植物的趋势,有助于进一步阐述SLE和排斥反应的发生机制。本研究的目的在于应用转录因子芯片技术,筛选在SLE和肾移植急性排斥反应中活性差异表达显著的TF,进一步探讨TF在SLE和肾移植急性排斥反应中的作用机制。研究材料与方法：1.10例取自健康自愿者外周血的单个核细胞为正常细胞对照组,15例取自SLE患者外周血的单个核细胞为SLE细胞实验组；取3例自肾肿瘤切除术中远离肿瘤组织且病理检查正常的肾皮质做为实验组织对照组,3例肾移植急性排斥反应患者肾穿标本做为肾移植急性排斥组。2.按照试剂盒说明抽提细胞核蛋白,bradford法用蛋白定量试剂盒定量蛋白浓度,定量后分装,-80℃冻存。3.标记所提取总核蛋白中的TF,浓缩标记样品,采用TranSignalTM Protein/DNA Array试剂盒进行TF芯片杂交。4.通过X胶片曝光,使用Genepix 4000B进行图像扫描,并转换为灰度TIFF格式的图片文件保存；采用ScanAlyze软件分析数据,分析结果导出,最终用EXCEL文件表示。5.电泳迁移率变动分析(EMSA)实验验证芯片结果。实验结果：1.所提取的细胞核蛋白经过定量检测符合芯片实验需求。2.SLE细胞实验组和对照组芯片研究结果发现92个TF差异表达显著,其中78个表达显著上调,14个表达显著下调。3.利用芯片比较分析急性组织排斥组和对照组TF表达,发现99个TF差异表达显著。其中95个表达显著上调,4个TF表达显著下调。4.筛选SLE细胞实验组和急性排斥反应组中差异表达显著的AP-1,Pbx1和MEF-2转录因子进行电泳迁移率变动分析实验研究,其结果均符合芯片分析结果。实验结论：1.微阵列芯片是有效的高通量分析SLE及肾移植急性排斥反应TF差异表达的研究手段。2.使用芯片筛选出92个在SLE患者中差异表达显著的TF；99个在肾移植急性排斥反应中差异表达显著的TF,证实TF在SLE和肾移植急性排斥反应中出现显著的差异表达。3.用电泳迁移率变动分析实验对所挑选的3个TF进行定量表达研究,其结果与芯片结果一致,证实了芯片结果的可靠性。电泳迁移率变动分析实验是检测单个TF表达水平的有效手段。TF有可能是SLE和肾移植急性排斥反应的新的有效生物标记。差异表达TF的研究有助于进一步了解SLE和肾移植急性排斥反应发生的分子机制。更多还原

【Abstract】 Systemic lupus erythematosus (SLE) is an unpredictable autoimmune disease involving multiple systems. Lupus nephritis is most common and most serious complication of it. At clinical statistics, SLE patient with renal damage is up to 95%, while the biopsy results are even more close to 100%. Since the glucocorticoid hormones and immunosuppressive agents have been used to treat SLE, the patient’s prognosis has been significantly improved, but the disease is still not optimistic about the treatment status. About 15% of the patients die and appear renal failure in five years. Meanwhile, Relapse during treatment is also facing a serious problem.Renal transplantation is the most ideal and oecumenical treatment of choice for end-stage renal disease (ESRD) patients. But transplantation rejection is still a strong risk factor for recipients of renal grafts. At present, the diagnosis of renal transplantation rejection can only be made by renal biopsy, which is costly, inconvenient and carries risks of complication.Clinical management of SLE and renal transplant patients would be improved if new rapid and reliable methods for detecting biomarkers of SLE and rejection were available. Despite a lot of work have been done, current clinical treatment of SLE commonly used glucocorticoid and/ or immunosuppressive drugs to suppress the body’s immune function. However, it is no target-oriented and lack targeting with facing immune functional disorder of SLE. At the same time, many patients are forced to withdrawal due to side effects of these drugs, leading to treatment interruption. Now investigators have not been able to use the results of rejection studies for early biomarkers form urine and peripheral blood of the patients in clinically.Transcription factor (TF) is a DNA-binding protein and also known as trans-acting factors. It can take place specificity interact with cis-acting element of eukaryotic gene promoter region-specific. Through them and with other relevant interactions between proteins, it is activating or inhibiting transcription. A transcription factor can control hundreds of genes’ expression, while the expression of a gene can also be regulated by a number of transcription factors. The structure and function of transcription factor defects are correlation to a number of human diseases (such as cancer and inflammation). Many transcription factors are still the role of drug targets.Novel biomarkers are readily, accessible and predict SLE and the fate of the transplant early and could help to further illustrate the mechanism of SLE and rejection. Our study Objective was to compare the levels of TFs expression in SLE and renal biopsies of acute rejection after renal transplantation between the normal samples and tried to reveal the mechanism of SLE and acute rejection after renal transplantation.Materials and Methods1. Fifteen cases of peripheral blood mononuclear cell of patients with SLE as experimental group, ten cases of peripheral blood mononuclear cell of healthy volunteers as normal control group; collected three biopsies of patients with acute rejection after renal transplantation as acute rejection group, three renal cortexes of tissue eumorphism patients as normal control group.2. Nuclear extracts were prepared using Nuclear Extract Kit according to the manufacturer’s instructions. Measure the protein concentration of each sample using a protein quantitation assay with Bradford method. Then it was quantitately subpackaged and stored in-80℃. Total RNA was extracted using trizol according to the manufacturer’s instructions and assessed RNA yield and quality by UV absorbance and denaturing agarose gel electrophoresis.3. The total TFs of nuclear extracts samples were labeling and concentrating and hybridization with TF array using TranSignalTM Protein/DNA Array microarray kit.4. Through X-film exposure, scan the slide using the Genepix 4000B.The images were saved as gray scale TIF files. Analyzed the data in ScanAlyze and saved the results as EXCEL files.5. Validated the results of TF array through EMSA.Results1. The quality of nuclear extracts was satisfied the demand of TF microarray experiment through the quantitative detection.2. In SLE, there were 92 TFs significant differential expression of which 78 up-regulated and 14 down-regulated.3. In acute rejection after kidney transplantation, there were 99 TFs significant differential expression of which 95 up-regulated and 4 down-regulated. 4. AP-1, Pbx1 and MEF-2 with a significant differential expressionin in SLE and acute rejection were selected for EMSA. It showed that the results were conformable to the result of array.Conclusions1. Microarray chip is the effective approach for the high-throughput analysis of TFs expression profile in SLE and acute rejection of renal transplantation.2. There were 92 TFs significant differential expression in SLE and 99 TFs significant differential expression in acute rejection after renal transplantation by Microarray chip. TFs were significant differential expression in SLE and acute rejection of renal transplantation.3.3 TFs were selected for EMSA. All results were conformable to the result of array which confirmed the reliability of Microarray chip results. EMSA was an effective approach for detecting expression level of the single TF.TFs may be the new biomarkers for SLE and acute rejection of renal transplantation.The study of differentially expressed TFs could help to further investigate the mechanism underlying the development of SLE and acute rejection of renal transplantation.更多还原