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Study on Expression of Proteome in Decidua of Recurrent Abortion Mice Affected by Shoutaiwan

Author: TanZhanWang
Tutor: LeiLei
School: Hunan University of Traditional Chinese Medicine
Course: Traditional Chinese Medicine
Keywords: Shoutaiwan Recurrent abortion Decidua Proteomics Two-dimensional electrophoresis Mass spectrometry
CLC: R285.5
Type: PhD thesis
Year: 2012
Downloads: 115
Quote: 1
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Abstract


Objective:To analysis the differential expression of decidua tissue proteins and effective mechanisms of recurrent abortion mice with Shoutaiwan,and explore the mechanism of Shoutaiwan in preventing miscarriage, in order to provide a theoretical basis for better clinical applications of Shoutaiwan.Method:1.The preparation of recurrent abortion models and intervention with Shoutaiwan:The abortion-prone CBA/J X DBA/2matings were established as the model of spontaneous abortion and the nonabortion-prone CBA/J×BALB/c matings were used as the model of normal pregnancy.The model of spontaneous abortion CBA/J X DBA/2of mice pregnant were randomly divided into four groups according to the sequence of pregnancy.From the1st day of pregnant,pregnant rats of every group are oral administration indifferent doses. On the14th days of pregnancy,the mice are killed and the embryo resorption rate was counted.2.Proteome analysis of decidua tissues in recurrent abortion mice:In the first place, the total proteins of mice decidua tissues at model group,normal group,low-dose Shoutaiwan group,middle-dose Shoutaiwan group and low-dose Shoutaiwan group, were separated by means of immobilized pH gradient(IPG)based2-DE and coomassie brilliant blue staining.The expression of differential protein spots was analyze by PDquest and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).The acquired Peptide mass fingerprints(PMFs)were used to search for matches in SWISS-PROT and NCBI databases with Mascot software.The functions of identified proteins were further analysed according to bioinformatics resources.3.Validating of differential expression proteins pots of HSP27, α-enolase,Transferrin and Annexin A2:In order to validate the reliability of the identified proteome analysis results,the expression of4differential protein spots of HSP27, α-enolase,Transferrin and Annexin A2in protein levels,which maybe correlated with recurrent abortion and Shoutaiwan intervention, was detected by immunohistochemical and Western blot analysis.Result:1.The models of embryo resorption rate are higher than the normal pregnancy group,compared with model group,low-dose Shoutaiwan can not significantly improve the model of spontaneous abortion in pregnant mice embryo resorption rate(P>0.05);Shoutaiwan high-dose group of pregnant mice absorption rate of the embryos were significantly decreased(P<0.01). 2.Proteome analysis of decidua tissues in recurrent abortion mice:The results showed that the well-resolved,reproducible2-DE patterns of mice decidua tissues of recurrent abortion,model group,normal group,low-dose Shoutaiwan group,middle-dose Shoutaiwan group and low-dose Shoutaiwan group were obtained.Analysis by PDQuest software, there are800protein points in every decidua respectivly.2-DE Gel analysis shows protein molecular weight concentrated mainly on10-100KDa,iso electric point was mainly in range of pH4.0-10.0.As the standard with the model decidua,proteins matching rate is83%between every decidua.Protein points of the differential expression are60.Up-regulation protein points over three times are28,and down-regulation protein points over three times are32.Through comparative proteome analysis of decidua tissues of model group,normal group,low-dose Shoutaiwan group,middle-dose Shoutaiwan group and low-dose Shoutaiwan group,30differential expression protein spots which maybe related to recurrent abortion and Shoutaiwan intervention were identified by MS.These differential expression proteins mainly refer to invasion of the blastocyst, blood vessel remodeling and cell apoptosis.3.Validating of differential expression proteins pots of HSP27, α-enolase,Transferrin and Annexin A2:Used the methods of Western blot and immunohistochemical,HSP27and α-enolase expression in model group are significantly higher than the normal pregnancy group(P< 0.01);Transferrin and Annexin A2expression,ara significantly lower than the normal pregnancy group at decidua tissues (P<0.01);Compared with model group,Shoutaiwan low middle high-dose group can decreased significantly HSP27and α-enolase expression in decidua tissues of pregnant mice(P<0.01);Shoutaiwan low middle high-dose group make Annexin A2expression in decidua tissues increased significantly(P<0.01);Compared with model group,Shoutaiwan high-dose group can increased significantly Transferrin in decidua tissues(P<0.01),Shoutaiwan middle low-dose group show no significant difference(P>0.05).These was consistent with our proteome analysis results.Conclusion:1.Shoutaiwan can decrease recurrent abortion mice absorption rate of the embryos significantly,and play a role in preventing miscarriage.2.The2-DE reference maps of decidua tissues of mice including model group,normal group,low-dose Shoutaiwan group,middle-dose Shoutaiwan group and low-dose Shoutaiwan group were established successfully.30differential expression proteins pots were identified.It indicated that recurrent abortion is a complicated process refer to diverse proteins participate and the comparison proteome analysis was a reliable method to sieve related proteins pots for recurrent abortion pathological changes.For several proteins pots expression of decidua tissues in recurrent abortion mice was regulated by Shoutaiwan,it provides contribution to the effect characteristic of multitarget.3.The expression of HSP27, α-enolase,Transferrin and Annexin A2in Protein levels with the methods of immunohistochemical and Western blot analysis was consistented with our proteome analysis results.Thus identificated the stability and reliable of proteome analysis.Shoutaiwan could down-regulate the expression of HSP27, α-enolase and up-regulate the expression of Transferrin,Annexin A2in protein levels,which may be one of the mechanisms of Shoutaiwans preventing miscarriage effect.

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