Comparative Proteomics Analysis Of Human Myometrium In No-labor And Labor Situation

The event of human parturition is also known to be a multifactorial,multiphase, multimolecular event in which several mechanisms actsimultaneously. Multimolecules are including multigenes and theirpassways and active proteins. To date, most researches about these genes&their passways and active proteins were focused on single or severalrelated genes or proteins, so it very hard to know the whole change ofproteins. Most research materials of parturition were limited in animalsand the lower segment of human myometrium, but their changes ofexpression can not replace the changes of corpus which undergoing theinitiative contraction of human parturition. Some new technologiessuch as comparative genome hybridization, microarray were adopted inthe researches on parturition of human myometrium, but it still can notprovide any information about the change level of protein. Anotherquestion is that information about protein modification, translocation andinteraction with other proteins or biomolecules can not be known frommRNA. Thus, if the information on the whole changed protein can beintegrated with the change information of mRNA, it will greatly help usto know more about human parturition.ChapterⅠ: Separation and identification of differentialexpressional proteins of human myometrium in no-labor and laborsituationObjective: To investigate comprehensively differential proteinsexpressive profiles in no-labor and labor situation in order to scan outlabor-associated proteins. Methods: Two-dimensional gel electrophoresis (2-DE) technologywas performed to separate the total proteins of corpus tissues and lowersegment sampled at two late stages of human pregnancy: full term not inlabor(NIL, 38-41 weeks amenorrhea) and full term in labor(IL, 38-41weeks amenorrhea), respectively. Then, Image Master 2D Platimumsoftware was used to analyze 2-DE images of same position in no-laborand labor situation, and the 24 selected differential expressional proteinswere identified by peptide mass fingerprint (PMF) mass spectrometrytechnology and the identified proteins have been classified in terms oftheir physiological function using information from SWISS-PROT andNCBI websites.Results:1. 2-DE patterns of the corpus and lower segment ofmyometrium in no-labor and labor situationThe well-resolved, reproducible 2-DE patterns of the corpus andlower segment of myometrium in no-labor and labor situations wereestablished. The total protein spots in corpus 2-DE profiles were counted622±14 and 654±34, and the correlation between two protein spots oftwo 2-DE patterns was 90.6％. The total spots in lower segment 2-DEprofiles of two labor situations were counted 432±14 and 441±24, andthe correlation between protein spots of two 2-DE pattems was 87.2％.2. Differentially expressive proteins in corpus and lower segmentof myometrium in no-labor and labor situation24 differential spots were identified by PMF. The differentialexpressional proteins could be divided into six main groups based ontheir functions:①proteins related to cellular structure;②calcium-bindingproteins;③chaperones;④energy and metabolic enzymes;⑤antioxygenenzyme;⑥proteins relative to signal transduction. 3. HSP27 expression of human myometrium in no-labor andlabor situationSeveral HSP27 spots which had identical molecular weight anddifferent isoelectric point were found in both 2-DE patterns of corpus andlower segment of myometrium in no-labor and labor situation, only 1point was significant difference significiance by Image 2D MasterPlatimum software analysis between two corpus profiles. The rest had nosignificiant difference.Conclusion:1. It was the first time to adopt human myometrium as researchmaterial for onset of human parturition by proteome technology, 20proteins were selected as labor-associated proteins in corpus ofmyometrium and 4 proteins were selected as labor-associated proteins inlower segment of myometrium2. Differential expression proteins could be classified into severalkinds. Calponin-1 and Transgelin were differentially expressed both incorpus and lower segment of myometrium, also different proteins wereidentified in both tissues of no-labor and labor situations, whichsuggested parturition was an course of multiproteins interaction.3. Different Annexin subunit was up-regulated in both corpus andlower segment in labor situation, which suggested signal transductionpassways might be different in corpus and lower segment at the onset ofhuman parturition.4. Vimentin was highly up-regulated in corpus in no-labor situation,which gave us a candidating target of tocolytic agent for prevention andtreatment of preterm labor.5. Transgelin was highly up-regulated in corpus at term inspontaneous labor, which suggested Transgelin might participate in parturition through signal passway.6. HSP27 was highly up-regulated in corpus at term in spontaneouslabor, which suggested HSP27 might participate in parturition through theinteraction with actin polymerization and chaperone.7. Several HSP27 spots which had identical molecular weight anddifferent isoelectric point were both found in 2-DE patterns of corpus andlower segment of myometrium of different labor situation. Therefore,specific post-translational modifications of HSP27 might play animportant role in human late pregnancy and parturition.ChapterⅡ: Differential expression of HSP27 in corpus ofhuman myometrium in no-labor and labor situation and therelationship with parturitionObjetive: In order to confirm the differential expressional HSP27protein was the true differential protein in corpus between two laborsituations.Methods: The tissues of corpus from human myometrium in laborand not in labor were collected in order to test the results of proteomicsanalysis. Half quantity RT-PCR technology, western-blot analysis andimmunohistochemistry technology were performed to determine thedifferential expressional levels of heat shock protein 27(HSP27).Results:RT-PCR: Expression of HSP27 rnRNA in corpus of NIL and ILwere 0.4801±0.0453和0.8320±0.0981, respectively. That in corpus ofIL were remarkable higher than NIL(P＜0.001).Western-blot: Expression of HSP27 protein in corpus of IL wasup-regulated obviously than NIL. Two straps were showed in both tissues.Immunohistochemistry: The optical density (OD) of expressionof HSP27 protein in corpus of myometrium in NIL and IL were 0.2290±0.0419, 0.6360±0.1083, respectively. The comparison between twolabor situation was significant difference.(P＜0.001).Conclusion: RT-PCR, Western-blot and Immunohistochemistrytechnology were usd to confirm the result of proteomics analysis.Fourmethods were proved each other to show that HSP27 was differentialexpression both at mRNA and protein level in corpus between two laborsituations. And specific post-translational modifications of HSP27 mightplay an important role in human late pregnancy and labor. HSP27, mightparticipate in human parturition by regulating actin cytoskeletondynamics and chaperone.ChapterⅢ: Differential expression of Transgelin in corpusof human myometrium in no-labor and labor situation and therelationship with parturitionObjctive: In order to confirm the differential expressionalTransgelin protein was the true differential protein in corpus betweentwo labor situations.Methods:The tissues of corpus from human myometrium inno-labor and labor situations were collected in order to test the resultsof proteomics analysis. Half quantity RT-PCR technology, western-blotanalysis and immunohistochemistry technology were performed todetermine the differential expressional levels of Transgelin.Results:RT-PCR: Expression of Transgelin mRNA in corpus of NIL and IL were 0.3526±0.0572, 0.9170±0.1483, respectively. That in corpus of ILwere remarkable higher than NIL(P＜0.05).Western-blot: Expression of Transgelin protein in corpus of IL wasup-regulated obviously than NIL.Immunohistochemistry:The optical density (OD) of expressionof Transgelin protein in corpus of myometrium in NIL and IL were0.1300±0.0100,0.3069±0.0968, respectively. The comparison betweentwo situation was significant difference (P＜0.05).Conclusion: RT-PCR,Western-blot and Immunohistochemistrytechnology were usd to confirm the result of proteomics analysis.Fourmethods were proved each other to show that Transgelin was differentialexpression both at mRNA and protein level in corpus between two laborsituations, which suggested Transgelin might participate in parturitionthrough signal transducation passway.Above all, with two dimensional electrophoresisi coupled massspectrometry technology, 24 selected differential expressional proteinswere identified in corpus and in lower segment of myometrium betweentwo different labor situation. The differential expressional proteins couldbe divided into six main groups based on their functions: proteins relatedto cellular structure,calcium-binding proteins, chaperones, antioxygenenzyme,energy and metabolic enzymes, and proteins relative to signaltransduction. Some differential expressed proteins such as Actin,S100A9proteins were known to be relative with human parturition; some mightbe new labor associated proteins, such as HSP27,Transgelin, and othershave not been linked to labor in corpus of human myometrium previously.RT-PCR,Western-blot and Immunohistochemistry technology were usdto determine the differential expressional level of partial proteins.The results were identical with the proteomics analysis both at mRNA andprotein level in corpus between two labor situation. Multimethods wereproved each other and drawn the same conclusion,which showed ourdatas were reliable and our proteomic analysis results were credible.These data are valuable for further study of the mechanism of humanparturition in myometrium and provide some new clues for searching newtarget proteins for prevention and treatment of preterm labor.