摘要
目的建立脑脊液双向差异凝胶电泳方法,寻找髓母细胞瘤脑脊液、室管膜瘤脑脊液与正常脑脊液三者之间的差异蛋白表达。方法选取3例已确诊髓母细胞瘤患者脑脊液等体积混合为髓母细胞瘤组(M);3例已确诊室管膜瘤患者脑脊液等体积混合为室管膜瘤组(E);3例正常脑脊液等体积混合为正常对照组(CON);分别采用10%TCA/冰丙酮沉淀法除盐提取蛋白;蛋白等量混合为内标组(S);将各组蛋白分别经花青染料Cy2、Cy3和Cy5标记,混合后进行双向差异凝胶电泳(2D-DIGE),分别在波长488nm、532nm和633nm激发光下扫描,所获得图像用DeCyder 5.0图像软件进行蛋白质表达差异分析。结果 DeCyder的胶内差异分析(DIA)模块显示胶1、胶2和胶3分别检测到1552、1547和1566个蛋白质点。DeCyder的生物学差异分析(BVA)模块显示表达量变化超过两倍的差异蛋白质点,在髓母细胞瘤组与正常对照组中有36个;室管膜瘤组与正常对照组中有71个;而髓母细胞瘤组与室管膜瘤组中有52个。其中仅在髓母细胞瘤中上调的蛋白质点有8个,仅在室管膜组中升高的蛋白质点10个。结论脑脊液双向差异凝胶电泳方法的成功建立,为髓母细胞瘤与室管膜瘤脑脊液中标志蛋白探寻研究奠定了基础。
Objective To establish the method of two-dimensional differential gel electrophoresis（2D-DIGE）and to find out the differentially expressed proteins in cerebrospinal fluid（CSF）between medulloblastoma,ependymoma and normal group.Methods 3cases of CSF of medulloblastoma（M）confirmed by postoperative pathology,then pooled by equal amount as M group;3cases of CSF of ependymoma（E）confirmed by postoperative pathology,then pooled by equal amount as E group;3cases of normal control CSF,then pooled by equal amount as Con group.The CSF samples were precipitated with cold 10% TCA/acetone.The internal standard comprised equal amounts of proteins extracted from three groups.Internal standardand proteins from the three groups were labeled with fluorescent dyes Cy2,Cy3 and Cy5and then pooled.2D-DIGE of labeled samples were run.2D-DIGE gels scanned with 488 nm,532nm and 633 nm wavelength laser were analyzed by DeCyde 5.0.Results Differential in-gel analysis（DIA）module of DeCyder indicated that there were 1552,1547 and 1566protein spots on gel 1,gel 2and gel 3respectively.Biological variation analysis（BVA）module of DeCyder indicated that when average ratio〉2.0or〈-2.0,there were differentially expressed protein spots 36 between M and Con,71 between E and Con,52 between M and E.Moreover,there were 8up-regulated spots only in M group and 10up-regulated spots only in M group.Conclusion The method of 2D-DIGE has been established succesfully,which provide a foundation for searching of the protein marker in the CSF of medulloblastoma or ependymoma.
出处
《中国实验诊断学》
2014年第8期1239-1242,共4页
Chinese Journal of Laboratory Diagnosis
基金
吉林省科技厅青年科研基金计划项目(项目编号20080133)
吉林省科技厅社发处计划项目(项目编号20050407-3)
关键词
髓母细胞瘤
室管膜瘤
脑脊液
双向差异凝胶电泳
Differential gel electrophoresis
medulloblastoma
ependymoma
cerebrospinal fluid
