Study The Placenta Tissue Protein Of Preelampsia

Preeclampsia,a common type of hypertensive disorder complicating pregnancyand a kind of idiosyncratic disease in pregnancy,has been considered a majorcontributor to the maternal and neonatal mortality and morbidity.The preciseetiopathogenesis of pre-eclampsia remains to be a subject of extensive research and ithas no ideal markers.Proteomics is a new technology,which includes proteinseparation,identification and bioinformatics.It has the virtue of high output and highsensitivity.Placenta plays a crucial role in the development of pre-eclampsia,whichcontact directly with basal deciduas and participate in immune tolerance andmaintaining pregnancy.We studied total proteins of placenta by using proteomicstechnology in this study,by comparing the differences of protein expression inplacenta of pre-eclampsia and normal pregnant women in hopes of finding associatedprotein of pre-eclampsia,so as to elucidate the pathogenic mechanism ofpreeclampsia and find out specific biomarkers.Objective:To investigate comprehensively differential proteins expressiveprofiles in placenta tissues of severely pre-eclampsia patients and normal pregnancywoman in order to scalp out pre-eclampsia-associationed proteins.In addition,find outsome antibodies to placenta antigen proteins in serum of pre-eclampsia .At last ,studythe differential expression of the antibodies in maternal peripheral blood ofpre-eclampsia patients to provide some clues for the pathomechanism and find outspecific biomarkers.Methods:1.The total proteins extracted from placenta tissues of preeclampsiapatients and normal controls were separated by means of two-dimensional gelelectrophoresis (2-DE);The first dimension was carried by IPG pH gradientisoelectric focusing system,the second dimension was separated by verticalSDS-PAGE,the late step was stained by silver or coomas brilliant blue forvisualization.Acquired gel images were analized and the spots were matched andcompared by PDQuest7.4 software (Bio-Rad).The statistical method was applized toevaluate the 2-DE results.2.Differentially expressed proteins spots in 2-DE maps were chosed and incised after digested in-gel by trypsin,and the candidate proteins were identified usingmatrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) .The peptide mass spectrometry maps and amino acid sequenceswere searched from SEQUEST databases.The functions and construction of theidentified proteins were searched by bioinformatics.3.Proteins from placenta tissues of preeclampsia patients were separated bytwo-dimensional PAGE.Then proteins were transferred to PVDF membranes.Serafrom the same patients with preeclapsia and normal controls were screened for IgGautoantibodies against the proteins by Western blot analysis.Finally massspectrometric analysis system was used to identify the constituents of the positiveproteins.Some antibodies to antigen proteins were further validated by ELISA amongserum.Results:1 The good reproducible results were acquired using the optimized2-DE technique.2 Compared with the normal placenta,18 protein spots with 2 fold of differencestatistically in PE placenta were found including 12 up-regulated and 6down-regulated protein spots.3 Fourteen proteins were identified by MALDI-TOF-MS,.Whereas levels ofFibrinogen beta chain precursor ,Vitamin D-binding protein,Tubulointerstitialnephritis antigen-like precursor,Fibrinogen gamma chain precursor ,Alpha-1-antitrypsin precursor ,Annexin A1,Alpha-enolase ,Keratin,typeâ… cytoskeletal 9,Keratin,typeâ…¡cytoskeletal 1,were upregulated in preeclapsia groupcompared with control group,we found a significant down regulation of Eukaryotictranslation initiation factor 5A-l,Ferritin light chain ,Transgelin-2,ATP synthasesubunit beta,Estradiol 17-beta-dehydrogenase 1 in preeclampsia group.4 Western blotting validated abnomal immunity in preeclampsia group.Acording 2-DE image find the position of placenta antigen.5 The peptide mass spectrometry maps and amino acid sequences were obtainedsuccessfully by digesting in-gel by trypsin and mass spectrometric analysissystem.Three placenta antigen were identified.There are antibody proteins againstFibrinogen beta chain,Heat shock 70kDa protein and Solution Structure of the Mitochondrial Ribosomal Protein or Keratin 9.6 The concentration of antibody proteins against fibrinogen beta chain and heatshock 70kDa protein in the preeclampsia maternal peripheral blood was higher thannormal pregnancy,which were paralleled with the results ofproteomic.Conclusion:1.2-DE pattern of placenta tissue from preeclampsia is differentfrom that of normal control group.The different proteins are invoulving in theimflammation,thrombosis,enzymes of glycolysis,physiological process of signaltransduction,cytoskeleton,cell survival and proliferation,cell apoptosis regulation,enzymes of energy metabolism and substances transport.2 Western blotting validated that there are abnomal immunity response againstplacenta proteins in preeclampsia group during the development of preeclampsia.Antibody proteins against Fibrinogen beta chain,Heat shock 70kDa protein andsolution Structure of the Mitochondrial Ribosomal Protein or Keratin 9 developeduring preeclampsia3 Antibody proteins against Fibrinogen beta chain,Heat shock 70kDa proteinmight play a crucial role for the pathogenesis.Perhaps they would be a candicatedbiomark of preelampsia.