Proteomics Study Of Uygur And Han Population With Primary Hyperuricemia In XinJiang

The large number of domestic and international epidemiological studies have shownthat the incidence of hyperuricemia (HUA) increase gradually with the improvement ofpeople’s living standards. HUA not only leads to gout and uric acid kidney diseases, butalso can cause insulin resistance, vascular lesions and glucose tolerance abnormal.There isclosely correlationship between HUA and metabolic syndrome (hypertention, arteryatherosclerosis, coronary heart disease, dyslipidemia, obesity and insulin resistance).Therefore it is very necessary and urgent to strengthen the study of hyperuricemia and toexplore its pathogenesis, prevent transforming to related diseases.The proteomics methodsbooming in recent years provide a powerful tool for the screening of a standard protein.In this study we have used proteomics to identify differentially expressed proteins ofUyghur and Han patients with HUA in comparison to normal subjects’serum.The validityof the differential protein expression in patient’s with HUA was further examined andconfirmed with Western-blot.From this study we want to found that the differentlyexpressed proteins in Uyghur and Han patients with HUA, to explore the proteins possiblyinvolve in the pathogenesis of hyperuricemia.And for applying the foundation for furtherstudying of interaction of environment factors and genetic factors on hyperuricemia.Objective:(1) Screen the differently expressed proteins in Uyghur and Han patients with HUAand normal population by2-DE.(2) Identify the differently expressed proteins by MALDI-TOF-MS/MS, select theproteins that possibly involve in the pathogenesis of HUA and transformation betweenHUA and metabolic diseases. Methods:(1) Collection for clinical data and serum samples15sera of Uyghur and Han patients with HUA were selected respectively as studygroups and the control groups were selected from health examination people in the FirstAffiliated Hospital.Blood sample was taken from fasting patients in the morning.Sera wasseparated and freezed.The serum is collected in the case of informed patients and healthyvolunteers, and all process meet the requirements of the Ethics Committee of the FirstAffiliated Hospital of Xinjiang Medical University(2) Two dimensional electrophoresis (2-DE)Total protein was extracted, purified and quantified, and then separated by2-DE.Different protein spots were stained by Coomassie Brilliant Blue, and the twodimentional electrophoregram was analyzed with the application of PDQuest7.3.0software to screen the different proteins.In order to ensure the reproducibility of theexperimental results, the above experiment was repeated three times under the sameconditions, different time.(3) Identification of proteins by MALDI-TOF–MS/MSprotein spots which displayed significant expressional differences (up-ordown-regulated by more than2.5times) were excised from the gel and sliced into sectionsabout1mm3.Samples were analyzed using MALDI-TOF-MS/MS.After MS analysis,peptides, which were different from matrix PMF (peptide mass fingerprint), were selectedand MS/MS (massspectrum/massspectrum) was achieved.(4) Database retrieval.The mass spectrum obtained from MALDI-TOF was searched using Mascotsoftware from NCBInr (www.matrixscience.com) and Swissprothttp (www.expasy.ch/sprot). The results were accordance in the two database and Total Ion Score C.I.%wasover95%were recogonized as successfully identified proteins.(5) Validation of differential expressed proteins.The levels of the proteins identified above (associated with HUA pathology) weremeasured in blood serum samples using Western-blot.Blood serums were obtainedrespectively from another30patients with HUA in Uyghur and Han and30normalsubjects in Uyghur and Han.Results:(1)2-D maps with a good reproducibility were obtained. After pretreatment, about650protein spots were respectively detected in2-dimensional electrophoresis profiles of Han and Uygur sera.By the quantitative analyse,there were8differential expressed protein spots in Han patients with HUA and10differential expressed protein spots in Uygur patients with HUA compared to controlgroups. Four of the total8proteins were up-expressed and other four proteins weredown-expressed.And nine of the total10proteins were up-expressed and other one proteinwas down-expressed in the serum.(2) Identification and verification of protein spots8PMFs were successfully identified by MALDI-TOF-MS/MS analyzing andretrieving NCBInr and Swissspot database.Complement3, haptoglobin, α1-antitrypsinwere up-regulated and apolipoprotein L1was down-regulated in Han patients as comparedwith control group. Complement3, complement4, haptoglobin, apolipoprotein A1wereup-regulated in Uygur patients as compared with control group.(3) Validation of complement3and haptoglobin by Western-blotComplement3and haptoglobin were validated by Western-blot and the results ofWestern-blot support the results of proteomic analysis.Conclusions:This study further validated that the proteomics techniques are powerful tools forscreening different proteins from patient’s serum.Our results revealed a significantlyincreased expression of Hp, C3and α1-AT in Han patients with HUA, and a significantlylowered level of APO L1in Han patients with HUA. And Complement3, complement4,haptoglobin, apolipoprotein A1are in higher level expression in Uygur patients’ sera thanin control.These proteins are correlated with immunization and lipid metabolism..Ourresults of comparative serum proteomics analysis might provide clues for furtherelucidating the role of these proteins in HUA. These differentially expressed proteins maybe involved in the HUA occurrence, development and the relationship betweenhyperuricemia and other metabolic syndrome diseases.