梅克尔憩室患儿差异性蛋白的筛选

Screening differentially expressed proteins of Meckel's diverticulum in children

目的采用双向电泳质谱分析筛选梅克尔憩室患儿和无肠道畸形儿童尸检小肠的差异蛋白。方法取无痛性便血梅克尔憩室患儿憩室顶端全层组织和无肠道畸形儿童尸检小肠组织。分别提取组织总蛋白、双向电泳分离、MALDI-TOF质谱分析、PubMed蛋白质序列检索,筛选得到黏膜差异蛋白。采用免疫组织化学法对憩室组38例患儿及对照组16例组织筛选出来的差异蛋白进行验证。结果筛选出11项表达差异大于3倍的蛋白,质谱分析及PubMed检索出5项蛋白得分高。其中,膜联蛋白A2(Anx-A2)、表皮生长因子受体(Egfr)低表达,细胞角蛋白9(Cyk9)和人类组织相关抗原I(MHC-I)在憩室异位黏膜高表达,血红蛋白被认为是憩室血管内残留。免疫组织化学显示Anx-A2在憩室基底层弱表达,对照组阳性表达;Cyk9在憩室壁黏膜层高表达,对照组阴性表达;MHC-I在憩室壁黏膜层阳性表达,对照组为弱阳性表达。结论Anx-A2低表达及Cyk9、MHC-I高表达可能与梅克尔憩室发病有关。

Objective To identify the differentially expressed proteins between Meckel's diverticulum and small intestine by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Methods Meckel's diverticulum tissue from children with painless bloody stool and small intestinal autopsy tissue were collected and cryopreserved. Total proteins were extracted and separated by 2-DE. The differentially expressed proteins between two groups were compared using image analysis software. The profiles of peptide mass fingerprint (PMF) were acquired after matrix assisted laser desortion/ionization time of flight mass spectrometry (MALDI-TOF-MS) and the proteins identified by searching the database of Pubmed. The expression of differential protein was confirmed by immunohistochemistry between Meckel's diverticulum tissue from 38 children and small intestine tissue from 16 normal controls. Results Comparative analysis of 2-DE maps revealed 11 differentially expressed proteins between two groups. Five differential proteins were further identified by mass spectrometry. Cyk9 and MHC-I were significantly up-regulated in experimental group than control group while Anx-A2 and EGFR showed significantly higher expressions in control group than in experimental group. Hemoglobin was considered to be intravascular residual in diverticulum. Immunohistochemistry showed that Anx-A2 was positively expressed in normal small intestine of control group and weakly expressed in diverticulum;Cyk9 negatively expressed in control group and highly expressed in diverticulum;MHC-I positively expressed in control group and strongly positively expressed in diverticulum. Conclusions Down-regulated Anx-A2, down-regulated MHC-I and up-regulated Cyk9 may be associated with the pathogenesis of Meckel's diverticulum.