【作者】 谭展望；

【导师】 雷磊；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 目的：分析寿胎丸对复发性流产小鼠蜕膜蛋白质组的影响,从分子水平探讨寿胎丸安胎的作用机制,为中医药防治复发性流产提供新的思路和理论依据。方法：1.复发性流产小鼠模型的制备及寿胎丸干预：CBA/J×BALB/C建立正常妊娠模型,CBA/J×DBA/2建立复发性流产模型,将复发性流产模型CBA/J×DBA/2孕鼠按怀孕先后顺序随机分为四组,分别为模型组、寿胎丸高剂量组、寿胎丸中剂量组和寿胎丸低剂量组,从妊娠第1天开始给药,孕14天后处死小鼠,计算各组胚胎丢失率。2.复发性流产小鼠蜕膜蛋白质组学研究：应用固相pH梯度(IPG)2-DE技术分别分离模型组、正常组、寿胎丸高、中、低剂量组小鼠蜕膜组织总蛋白质后,经考染显色、PDQuest图像分析、识别差异表达的蛋白质点,应用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF-MS)分析得到肽质量指纹图谱(PMF),再经Mascot软件搜索SWISS-PROT和NCBI蛋白质数据库鉴定蛋白质,并利用生物信息学资源对所鉴定蛋白质的功能进行分析。3.差异蛋白质HSP27、α-enolase、Transferrin、Annexin A2结果验证：采用蛋白印迹和免疫组化方法对4个可能与复发性流产及寿胎丸作用相关的差异蛋白质HSP27、α-enolase、Transferrin、Annexin A2从蛋白水平的表达情况验证蛋白质组学鉴定结果的可靠性。结果：1.复发性流产小鼠模型的制备及寿胎丸干预：小鼠模型组与正常组相比,胚胎丢失率明显升高,差异有显著统计学意义(P<0.01)；寿胎丸高、中剂量组与模型组相比,胚胎丢失率均降低,差异有显著统计学意义(P<0.01)；寿胎丸低剂量组未能明显改善复发性流产小鼠胚胎丢失率,与模型组相比差异无统计学意义(P>0.05)。2.复发性流产小鼠蜕膜蛋白质组学研究：建立了模型组、正常组、寿胎丸高、中、低剂量组小鼠蜕膜组织的2-DE图谱。PDQuest软件的分析结果显示,每张2-DE图谱约有800个蛋白质点,蛋白质分子量(Mw)主要集中在10-100KDa范围内,等电点(pI)主要分布在pH4.0-10.0之间。以模型组为参照,其余各组蛋白点与其匹配率为83%,表达差异3倍以上的蛋白点有60个,其中上调的点有28个,下调的点有32个。通过比较模型组、正常组、寿胎丸高、中、低剂量组双向凝胶电泳图谱的差异,发现并鉴定了30个可能与复发性流产及寿胎丸干预相关的蛋白质,这些蛋白质的功能涉及绒毛外滋养细胞的侵袭、蜕膜组织血管的重建及蜕膜细胞的凋亡等。3.差异蛋白质HSP27、α-enolase、Transferrin、Annexin A2结果验证：蛋白印迹和免疫组化检测结果显示,小鼠模型组与正常组相比,蜕膜HSP27、α-enolase表达明显升高,Transfern、Annexin A2表达明显降低,差异有显著统计学意义(P<0.01)；寿胎丸高、中、低剂量组与模型组相比,蜕膜HSP27、α-enolase表达均降低,AnnexinA2表达均升高,差异有显著统计学意义(P<0.01)；寿胎丸高剂量组与模型组相比,蜕膜Transferrin表达升高,差异有显著统计学意义(P<0.01)；寿胎丸中、低剂量组与模型组相比,蜕膜Transferrin表达差异无统计学意义(P>0.05)。证实各实验组中HSP27、α-enolase、 Transferrin、Annexin A2于蛋白表达水平和2-DE结果相一致。结论：1.寿胎丸能够降低复发性流产小鼠模型的胚胎丢失率,具有显著的安胎作用。2.成功建立了模型组、正常组、寿胎丸高、中、低剂量组的双向凝胶电泳(2-DE)图谱,鉴定了30个差异表达的蛋白质,提示复发性流产是一多种蛋白质参与的复杂过程,小鼠蜕膜组织的比较蛋白质组学分析是筛选复发性流产病变相关蛋白质的可靠方法之一；寿胎丸可调节复发性流产小鼠蜕膜组织多种蛋白质的表达,提示该复方具有多靶点的作用特点。3.HSP27、α-enolase、Transferrin、Annexin A2的蛋白印迹和免疫组化验证结果与蛋白质组学分析结果一致,从一定程度上证明了蛋白质组学实验方法的稳定性及其结果的准确性；寿胎丸通过下调复发性流产小鼠蜕膜组织HSP27、α-enolase蛋白的表达,同时上调Transferrin、Annexin A2蛋白的表达,从而实现维持妊娠,降低胚胎丢失率,这可能是其安胎的作用机制之一。更多还原

【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.更多还原