Urine Proteomics Research Of IgA Nephropathy By Excessive Or Deficiency Syndrome And Its Clinical Significance

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.