【作者】 祁爱蓉；

【导师】 李顺民；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 IgA肾病(IgA nephropathy,IgAN)是临床血尿患者中最常见的肾小球疾病,与免疫异常高度相关。目前尿液蛋白质组学已经应用于医学领域的研究,包括肾脏病学研究。但尿液蛋白质组学应用于IgAN的极早期中医证侯分型、不同病理类型的鉴别诊断方面的研究尚未见报道。本研究收集IgAN虚证、实证患者的尿液用高速离心、超滤管浓缩、透析除盐以及乙腈萃取等方法提取一定浓度的尿蛋白,利用双向电泳技术分离不同等电点和分子量的蛋白,并通过胶内酶切联合MALDI-TOF-MS质谱分析,对获得的肽片段信息进行蛋白数据库检索,同时结合其临床资料、文献研究等,对鉴定出的蛋白质包含的生物学信息和临床意义进行初步分析。目的:比较三组观察对象(第一组:正常与阴性对照组,即N与C组;第二组:IgAN虚证与实证组,即D与E组;第三组:IgAN蛋白尿阳性与局灶节段硬化性肾小球肾炎组(FSGS),即A与F组)其尿液差异蛋白图谱,并利用质谱技术鉴定相关蛋白,并对其生物学信息和临床意义进行分析,具体如下:1通过文献研究了解IgAN的中、西医诊疗进展及蛋白组学研究动态;2比较IgAN虚、实证患者临床生化及免疫学指标的差异;3探讨IgAN与FSGS患者;IgAN虚、实证患者:健康人与IgA肾病阴性对照患者三组观察对象的尿液差异蛋白图谱,为临床分析不同病理类型肾小球肾炎提供实验依据;4比较IgAN虚、实证患者尿液差异蛋白图谱,并通过质谱分析,寻找功能蛋白,分析其临床相关性。方法:在文献研究的基础上,优化预实验方案,并对上述三组观察对象利用双向电泳技术,分离出组内差异蛋白点,对差异明显的蛋白点进行胶内酶切,质谱分析,获得肽质量指纹图,通过相关蛋白质数据库进行检索,鉴定出蛋白质,并将鉴定出的蛋白质输入Harvester蛋白搜索框(http://harvester.embl.de)和SWISS-Prot数据库可以得到相关蛋白生物信息学数据,并进一步在SWISS-Prot和EMBL(Harvester)数据库查询,获得蛋白质鉴定和识别、相似性分析、模式查询、翻译后预测、一级结构分析、二级结构预测、三级结构、跨膜区的预测等相关数据。具体如下:1文献研究:通过查阅大量文献,总结并分析IgAN的中、西诊疗进展及蛋白组学研究动态,提出IgAN中医蛋白组学研究假说。2实验研究:①预实验:在上述假说的基础上,参考相关蛋白组学文献提供的方法和经验,拟定预实验方案,筛选本实验的观察对象,确定标本留取方法、尿液蛋白样品的制备步骤,通过蛋白定量和SDS-PAGE小胶的情况,选取提取成功的尿液样品进行预实验。②正式实验:选取符合条件的观察对象60例,分为正常对照组(Normal,N组)、IgA肾病蛋白尿阴性对照组(Control,C组)、FSGS组(F组)、IgA肾病蛋白尿组(A组)、IgA肾病虚证组(Deficiency,E组)、IgA肾病实证组(Excessive,F组)各10例,(其中有14例患者为重复观察对象,具体见后续临床研究资料)。所有观察对象均取晨尿约20-200ml(N与C组样品为200ml,其余组为20ml),混匀后取原等量样品,-80℃冻存备用。临用前每10ml尿中加入6uL 100mM PMSF(苯甲基磺酰氟)防蛋白裂解。通过高速超滤及针头式过滤器,除去细胞核碎片及细菌,过滤后的尿液通过YM-3超滤管(3000Da)及透析袋进一步浓缩和除盐,然后用适量蛋白裂解液(成分:40MmTris,7M urea,2M thiourea,4%CHAPS,1.5%DTT,1.5%IPG buffer)复溶,用Brandford法测定蛋白浓度,蛋白上样量约200-400μg。尿蛋白经乙腈沉淀、除盐和离心管超滤浓缩后进行等电聚焦(IEF),聚焦后的胶条平衡后转移到垂直电泳槽进行SDS-PAGE电泳。以银染对SDS-PAGE胶进行显色,以perkin Elmer type型凝胶扫描仪扫描,以imageMaster 5.0分析软件进行分析差异表达的蛋白质点。并对差异蛋白点取点、酶解后进行MALDI-TOF-MS质谱分析,获得肽质量指纹图,通过相关蛋白质数据库进行检索分析。3临床研究:对上述60例观察对象,结合其临床病理分型、血常规白细胞总数、肾功能、补体C3水平、尿TGF-β、尿红细胞形态分析等临床资料,总结它们与差异蛋白信息的相关性。结果:通过预实验我们观察到:对于18cm pH3-10的线性胶条而言,合适的上样量为200ug,聚焦条件为60000伏小时。通过正确合理的研究,我们成功获取了IgA肾病、FSGS肾病、健康人、IgA肾病蛋白尿阴性对照组、IgA肾病虚、实证组等观察对象的尿液2-D图谱。ImageMaster 5.0软件进行图像分析,IgA肾病与FSGS肾病组(A组与F组)的3次凝胶的平均蛋白质点数分别为455和784;IgA肾病虚证与实证组(E组与D组)平均蛋白质点数分别为489和568;健康人与IgA肾病蛋白尿阴性对照组(N组与C组)平均蛋白质点数分别为282和350;其中A组与F组有明显差异的蛋白点22个,鉴定有意义的蛋白3个;E组与D组有明显差异的蛋白点35个,鉴定有意义的蛋白18个;N组与C组有明显差异的蛋白点20个,鉴定有意义的蛋白2个;具体分析显示:蛋白免疫功能异常可能为参与部分IgA肾病患者病情反复导致原发病加重的可能因素。IgA肾病实证患者,其尿中HSPG2;IGKC:HBA1;LOC554223:TCRP;complex-forming glycoprotein HC;immunoglobulin heavy chain variableregion等蛋白均有差异性表达。1文献研究:文献研究表明:中医药蛋白质组学技术相对中医精髓理论“辨证论治、整体观”而言,有着惊人的相似,即:执行人体生命活动的功能体是一个统一的整体,蛋白质组正是以一组蛋白的表达谱来共同呈现某种功能的。在这种趋势下,我们提出IgA肾病中医蛋白组学研究假说是合理的。2实验研究:分三组进行2-D电泳、质谱分析、相关数据库搜寻后发现,有意义的蛋白点分别是:相关研究结果显示:第一组差异蛋白点35个,鉴定有意义蛋白点18个,它们是:Chain B;Chain A;albumin preproprotein:IgG kappa chain(IgG Kappa链);specific heparan;hemoglobin alpha 1 globin chain(人类血红蛋白1抗体);T-cell receptor germlineJ-alpha RP region(TCR-β);complex-forming glycoprotein HC(人类糖蛋白);immunoglobulin heavy chain variable region(免疫球蛋白重链可变区);PR02044LOC554223protein;PR02675;basement membrane-specific heparan;alpha-1-micro globulin/bikunin preproprotein(NP001624);Chain A,Crystal Structure OfThe Fab Fragment Of A Human Monoclonal Igm Cold Agglutinin(1QLRA);anti-Entamoeba histolytica immunoglobulin kappa light chain(BAA82103);immunoglobulin kappa light chain VLJ region(BAC01750);Ig kappa chain NIG93;precursor(JE0243)。第二组差异蛋白点22个,鉴定有意义蛋白点3个它们是:PGBM;inhibitot BrCNfragmentⅡ,alphal prote;Chain A,Deoxy Rhb1.2(Recombinant Hemoglobin)第三组差异蛋白点20个,鉴定有意义蛋白点2个它们是:HSPG2;typeⅡkeratinsubunit protein3临床研究:IgAN患者虚证、实证组(即E与D组)比较:D组患者,血白细胞数与24h尿蛋白定量水平(CSF)、尿红细胞形态分析中红细胞总数,均明显高于E组,差异有统计学意义,P＜0.05:但其补体C3水平、平均血红蛋白含量(HGB)、血清肌酐(Scr)、尿素氮水平(BUN)比较,两组差异无统计学意义,P＞0.05。正常人与IgA阴性对照组(即N与C组)比较:N组患者24h尿蛋白定量水平(CSF)、尿TGF-β水平均高于C组;补体C3水平低于C组,差异有统计学意义,P＜0.05;而且C组患者尿红细胞形态分析中红细胞总数及形态均明显与N组不同,其图例参见附表。但其血白细胞总数、平均血红蛋白含量(HGB)、血清肌酐(Scr)、尿素氮水平(BUN)比较,两组差异无统计学意义,P＞0.05。IgAN蛋白尿阳性与(FSGS)组(即A与F组)比较:A组患者,其尿液红细胞总数高于F组;但补体C3水平、24h尿蛋白定量水平(CSF)均明显低于F组,差异有统计学意义,P＜0.05;但血白细胞总数、血清肌酐(Scr),尿素氮水平(BUN),尿TGF-β水平均无明显统计学意义,P＞0.05。结论:在结合文献研究及IgA肾病虚、实证患者的尿液差异蛋白图谱实验研究的基础上,我们可以得出以下结论:1、IgA肾病实证患者,其尿中IgG kappa chain;hemoglobin alpha 1 globinchain;T-cell receptor germline J-alpha RP region;complex-forming glycoprotein HC;immunoglobulin heavy chain variable region蛋白均有明显差异性表达,因此可作为实证患者病情判断的指标之一。其中,HSPG2和T-cell receptor germlineJ-alpha RP region蛋白的表达异常可能与临床方面此病情反复发作常有上呼吸道感染或扁桃体炎史相关,此外本研究也提示,与感染诱因相关的T-cell receptor germline J-alpha RP region蛋白同样在IgA肾病患者虚、实证的转化中起比较关键的作用。2、而且通过其他二组即:N与C组;A与F组,作对照,其显示的差异点和相关蛋白质鉴定,进一步印证了此实验的可靠性。3、结合临床资料分析,IgA肾病患者虚证(E组)、与实证组(F组)比较:基于其血白细胞总数、24h尿蛋白定量水平(CSF)、尿红细胞形态分析、补体C3水平、血清肌酐(Scr),尿素氮水平(BUN)比较的差异,提示:IgA肾病实证患者其尿蛋白定量及白细胞平均水平均较虚证患者为高,可能与疾病病情变化有关,由于机体免疫功能异常,激活细胞或体液免疫系统,引起新一轮的免疫应答,表现在中医证侯分型方面,由虚证向虚实夹杂或实证转化。更多还原

【Abstract】 IgA nephropathy(IgAN) is the most common glomerular disease among Clinical hematuria patients,and highly relevant to immune abnormalities.At present,the urine has been applied to proteomics research in the field of medicine,including nephrology research.However,IgAN urine proteomics applied to the very early TCM syndrome typing and different types of differential diagnosis of pathological research has not been reported.The Deficiency or excessive syndrome of IgAN patients were observed,the urine of them were collected,and were used with high-speed centrifugation, ultrafiltration and tube concentration,then were dialysised and desalted with acetonitrile extraction methods to a suitable range of concentration, After doing that,the samples were used two-dimensional gel electrophoresis separation for different isoelectric point and molecular weight protein,and digested by trypsin and combined MALDI-TOF-MS mass spectrometry analysis of peptide mixtures obtained by the quality of data related protein database search,combined with its clinical data,literature research identified prot ein contained in bioinformatics and clinical significance of the preliminary analysis.ObjectiveTo establish the two imensional gel electrophoresis and explore the expressions of differential proteins between three groups,(N and C groups,the normal and control groups;D and E groups,The Deficiency or excessive syndrome of IgAN group;A and F groups,IgAN and FSGS groups),as follows:1.Understanding through literature IgAN of Chinese and Western medical progress and proteomics research.2.Comparison of IgAN with syndrome of deficiency or excessive patients with evidence of clinical and immunological indicators of the students.3.To observe the objects of urine protein pattern differences IgAN and FSGS patients;IgAN with syndrome of deficiency or excessive patients;healthy people and the negative control group of IgAN,and discussion clinical analysis of different pathological types of glomerulonephritis and provide experimental basis.4.Comparison the different protein patterns of IgAN with syndrome of deficiency or excessive patients,and through mass spectrometry analysis, looking for functional proteins,analysis of its clinical relevance.Methods:Studies in the literature,based on the optimization of pre-pilot program, and the above-mentioned three groups of subjects using two-dimensional gel electrophoresis,isolated from group differences in protein spots on the obvious differences in protein-point line trypsin digestion,mass spectrometry analysis,was peptide mass fingerprinting through relevant protein database search analysis identified proteins,Will be identified protein input protein database search box can be associated protein bioinformatics data.And identified the proteins Bioinformatics preliminary analysis.Of this protein in the database query,to obtain protein identific ation and recognition,similarity analysis,model inquiry,post-translational prediction,primary structure analysis,secondary structure prediction, tertiary structure,prediction of transmembrane region and other related data. As follows:1 One literature study:through access to a large number of documents, summary and analysis of IgAN in Chinese and Western medical progress and proteomics research,the IgAN group study of Chinese medicine protein hypothesis.2 Experimental Study:①Pre-experiment:in the above-mentioned hypothesis on the basis of the reference literature related to the provision of proteomics methods and experiences,to develop pre-pilot program,the selection of the experimental observation of the object to determine the method of collecting specimens, urine protein sample preparation steps Protein quantification and SDS-PAGE mini gel situation,select the successful extraction of the urine samples were pre-experiment.②formal experiment:select eligible observed 60 cases of objects, divided into normal control group(Normal,N group),IgA nephropathy proteinuria negative control group(Control,C groups),FSGS group(F group), IgA nephropathy proteinuria group(A group),IgAN group Deficiency(Excessive, E group),IgAN empirical group(Deficiency,F group) of the 10 cases,(which has 14 cases of patients with repeated observations for the object,see the specific follow-up Clinical research data).All obser red objects are taking morning urine of about 20-200ml(N and C samples for the 200ml,the remaining group,20ml),after mixing samples from the original contour,-20℃cryopreservation standby.Pro per 10ml of urine before adding 6uL 100mM PMSF anti- protein cleavage.Ultrafiltration and high-speed needle-type filter,remove the nuclear debris and bacteria,filtered urine ultrafiltration through YM-3 tubes(3000Da) and dialysis bag further enrichment and desalting,and then use the appropriate protein lysis buffer(Composition:40Mm Tris,7M urea,2M thiourea,4%CHAPS, 1.5%DTT,1.5%IPG buffer) Repeated dissolution,Brandford method with protein concentration,The urinary protein of these odserved peoples was isolated by Acetonitrile precipitation,treated to remove salts and concentrate by Centrifugal Ultrafiltration tube.The rehydrated IPG strips containing protein samples were isoelectric focusing(IEF),after IEF,the equilibrated strips are transferred to the vertical SDS gel.The silver stain ed gels were scanned by proxpress 2-D proteomics imaging system,perkin Elmer type.after following imageMaster 5.0 analysis,the differentially expressed protein spots were found,according to 200-400μg of the sample volume has been carried out isoelectric focusing and second to the SDS-PAGE electrophoresis, and differences in protein-point check point,after digestion MALDI-TOF-MS-MS analysis,peptide mass fingerprint obtained by the relevant protein database search analysis.3 clinical studies:by collecting 60 cases of the above experiment for collecting specimens of patients with IgAN time clinical data,analysis of its clinical pathological type,serum WBC,renal function and urinary TGF-β, urine red blood cell morphology analysis,serum complement C3 biochemical indicators of the level of immune proteins and differences of information related to each other.Results:Using the proper method stated above,satisfactory 2-D maps of urinary protein was obtained and the preliminary analysis results were reported. The reasonable sample load and Volt-hours of urinary protein were about 200ug and 60000 volt-hours for the 18 cm pH3-10 IPG strips.A total of 455 protein spots were visualized in the patient with IgAN patients,784 protein spots in the patient with FSGS;A total of 489 protein spots were visualized in deficiency syndrome of IgAN,568 protein spots in the patient with excessive group;A total of 350 protein spots were visualized in the patient with control groups,282 protein spots in the patient with normal people;between A group and Group F,there are 22 protein spots significant differencets,succeedly identification significant 3 protein spots;E group and D group,there are 35 protein spots significant differencets,succeedly identification significant 18 protein spots;N group and C group there are 20 protein spots significant differencets,succeedly identification significant 2 protein spots.Specific analysis revealed:protein,immune dysfunction may be involved in some IgAN patients with recurrent primary disease leading to the possibility of aggravating factors.Positive patients with IgAN,the urinary HSPG2;IgG kappa chain;hemoglobin alpha 1 globin chain;T-cell receptor germline J-alpha RP region;complex-forming glycoprotein HC;immunoglobulin heavy chain variable region proteins were highly expressed.1 One literature study:Literature study shows that:The Chinese medical proteomics technology relative essence of traditional Chinese medicine theory "syndrome diff erentiation,the overall concept" is concerned,have a surprisingly similar, namely:the implementation of the functional activities of human life,is a unified whole,the protein group is a group of protein expression profiling to show some common features.In this trend,we propose IgAN TCM proteomics research hypothesis is reasonable.2 Experimental:Divided into three groups for 2-D electrophoresis,mass spectrometry analysis,relational database search revealed that significant protein spots are:relevant research findings show:Difference between the first group of 35 protein spots,identified 18 significant protein spots,which are:Chain B;Chain A;albumin preproprotein; IgG kappa chain(IgG Kappa chain);specific heparan;hemoglobin alpha 1 globin chain(human hemoglobin 1 antibody);T-cell receptor germline J-alpha RP region(TCR-β);complex-forming glycoprotein HC(human glycoprotein); immunoglobulin heavy chain variable region(immunoglobulin heavy chain variab le region);PRO2044 LOC554223protein;PRO2675;basement membrane-specific heparan;alpha-1-mic ro globulin / bikunin preproprotein(NP001624);Chain A,Crystal Structure Of The Fab Fragment Of A Human Monoclonal Igm Cold Agglutinin(1QLRA);anti-Entamoeba histolytica immunoglobulin kappa light chain(BAA82103);immunoglobulin kappa light chain VLJ region(BAC01750); Ig kappa chain NIG93 precursor(JE0243).The second group differences in 22 protein spots to identify meaningful proteins they are 3 points:PGBM;inhibitor BrCN fragmentⅡ,alphal prote; Chain A,Deoxy Rhb1.2(Recombinant Hemoglobin).The third group differences in 20 protein spots to identify meaningful proteins they are 2 points:HSPG2;type iI keratin;subunit protein.3 Clinical Research:Deficiency and excessive syndromes of IgAN,(that is,E and D group),the WBC counts and 24h urinary protein levels(CSF),urine red blood cell morphology analysis,the total number of red blood cellsof D group,were significantly higher than the E group,There were significant differences, P<0.05;but the level of complement C3,serum creatinine(Scr),blood urea nitrogen level(BUN) compared the two groups were no significant difference, P>0.05.Normal and IgA-negative control group(that is,N and C) Comparison:N group of patients with 24h urinary protein levels(CSF),urine TGF-βhigher than that of C group;complement C3 were lower than the C group,the difference statistically significance,P <0.05;and C group patients with urinary red blood cell morphology analysis,the total number of red blood cells and form with the N group were significantly different,the legend see Table.But the total number of leukocytes,the average hemoglobin content(HGB),serum creatinine(Scr),blood urea nitrogen level(BUN) compared the two groups was no significant difference,P> 0.05.IgAN with positive proteinuria(FSGS) group(that is,A and F) Comparison: A group of patients,their urine is higher than the total number of red blood cell group F;but complement C3 levels,24h urinary protein levels(CSF) were significantly lower than F group,the difference was statistically significant, P<0.05;but the total number of leukocytes,serum creatinine(Scr),blood urea nitrogen level(BUN),urinary TGF-βlevels,urine protein electroph oresis the distribution of statistical significance was no significant,P＞0.05.Conclusion:The combination of literature research and IgAN is true,positive differences in the urine of patients with protein atlas based on experimental studies,we can draw the following conclusions:1.IgAN in patients with evidence of its urinary IgG kappa chain; hemoglobin alpha 1 globin chain;T-cell receptor germline J-alpha RP region; complex-forming glycoprotein HC;immunoglobulin heavy chain variable region proteins were highly expressed,it can be Empirical patients judged as one of the indicators.One,T-cell receptor germline J-alpha RP region abnormal protein expression may be related to the clinical aspects of this condition often recurrent upper respiratory tract infection or tonsillitis relevant history,in addition to this study also suggests that incentives associated with the infected T-cell receptor germline J-alpha RP region the same protein in patients with IgAN is true,the transformation of empirical play more crucial role.2.but also through other two groups namely:N and C groups;A and F groups, as control,which showed differences in point and the identification of related proteins,and further confirms the reliability of this experiment.3.combined with clinical data analysis,IgAN in patients with Deficiency (E group),and empirical group(F group) comparison:on the basis of their WBC,24h urinary protein levels(CSF),urine red blood cell morphology analysis, complement C3 levels,serum creatinine(Scr),blood urea nitrogen level(BUN) compared the difference between prompt:IgAN in patients with evidence of their urinary protein and interleukin-average higher than those in patients with deficiency may be related to changes in disease-related illness,due to immune function abnormal activation of cells or humoral immune system,trigger a new round of the immune response,manifested in the TCM syndrome typing,from deficiency to the actual situation or a mixture of positive transformation.更多还原