早期诊断唐氏综合征的母体血清蛋白新标志物筛选与初探验证

Screening and preliminary validation of new protein markers in maternal serum for early diagnosis of Down syndrome

目的探讨经筛选与验证的唐氏综合征（DS）相关特异性母体血清蛋白新标志物的临床应用价值。方法选取确诊为DS胎儿的孕中期（14—20周）母体血清7份（DS组）和同期正常胎儿母体血清7份（正常对照组）行二维凝胶电泳（2-DE），建立DS胎儿母体血清蛋白表达差异图谱，提取差异表达蛋白质点进行质谱分析。免疫印迹法（WB）验证部分差异蛋白的表达差异。采用近似t检验比较分析2组间相应蛋白条带的吸光度（A）值差异。结果经2-DE和差异蛋白图谱分析，在DS组母体血清中共发现表达差异1．5倍以上的蛋白质点29个，其中表达上调的19个，表达下调的10个。表达差异2倍以上的8个蛋白质点经质谱分析，从美国国立生物技术信息中心（NCBI）蛋白质序列数据库中搜索为dGTP酶（dGTPase）和B2．糖蛋白I（132．GPI）等12个蛋白质分别与之匹配的可能性较大（概率得分〉66分）。WB证实GTPase和B2-GPI的A值在Ds组分别为21567．0±3009．4和22097．0±3958．9，在正常对照组分别为3957．7±250．9和1799．7±105．5，dGTP酶和β2-GPI在Ds胎儿母体血清中比正常对照组显著高表达（t’dGTPase=-17．66，t’β2-GPI=-14．83，P均〈0．0001）。结论2-DE和质谱技术是初步筛选DS相关特异性母体血清中新蛋白标志物的有效方法，经WB验证的dGTPase和β2-GPI可能是筛选Ds的新标志物。

Objective To assess the clinical application values of the protein markers associated with Down syndrome （DS）in maternal serum which were screened and identified. Methods Seven maternal serum samples with DS fetus （DS group） and 7 maternal serum samples with normal fetus （control group） in the second trimester were separated by two-dimensional gel electrophor6sis （2-DE）. The differentinal expression profile of proteome in maternal serum from DS group was established. The differentially expressed proteins were screened by mass spectrometry （MS） and some proteins were verified by Western blotting （WB）. Results Twenty-nine proteins were discovered to be differentially expressed by more than 1.5 folds in maternal serum from DS group, among which 19 proteins were up-regulated and 10 proteins were down-regulated. Eight proteins displayed 2 or more folds changes in maternal serum from DS group were identified by MS and possibly matched with 12 proteins in Ameracan National Center of Biotchnology Information （NCBI） protein sequence database, such as dGTPase and Beta2-Glycoprotein I （ β2-GPI）, etc. The results of WB showed that the mean a values of dGTPase and β2-GPI were 21 567.0± 3009.4 and 22 097.0 ± 3958.9 in the DS group, 3957.7 ± 250. 9 and 1799. 7 105.5 in the control group respectively, which presented that the expression of dGTPase and β2-GPI significantly increased in DS group （ t＇dGYPase = -17.66, t＇ β2-GPI：=- 14. 83,P 〈 0. 0001 ）. Conclusions 2-DE and MS are effective methods for preliminary identification of protein markers associated with DS in maternal serum, dGTPase and β2-GPI verified by WB laid a solid fundation for further screening new biologic markers for early diaglosis of DS.