CAJ | 학술논문

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488 nm、532 nm 和633 nm 激发光下扫描，所获得图像用 DeCy-der 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。
목적：건립뇌척액쌍향차이응효전영방법，심조수모세포류뇌척액、실관막류뇌척액여정상뇌척액삼자지간적차이단백표체。방법선취3례이학진수모세포류환자뇌척액등체적혼합위수모세포류조（M）；3례이학진실관막류환자뇌척액등체적혼합위실관막류조（E）；3례정상뇌척액등체적혼합위정상대조조（CON）；분별채용10％TCA/빙병동침정법제염제취단백；단백등량혼합위내표조（S）；장각조단백분별경화청염료 Cy2、Cy3화 Cy5표기，혼합후진행쌍향차이응효전영（2D-DIGE），분별재파장488 nm、532 nm 화633 nm 격발광하소묘，소획득도상용 DeCy-der 5．0도상연건진행단백질표체차이분석。결과DeCyder 적효내차이분석（DIA）모괴현시효1、효2화효3분별검측도1552、1547화1566개단백질점。DeCyder 적생물학차이분석（BVA）모괴현시표체량변화초과량배적차이단백질점，재수모세포류조여정상대조조중유36개；실관막류조여정상대조조중유71개；이수모세포류조여실관막류조중유52개。기중부재수모세포류중상조적단백질점유8개，부재실관막조중승고적단백질점10개。결론뇌척액쌍향차이응효전영방법적성공건립，위수모세포류여실관막류뇌척액중표지단백탐심연구전정료기출。
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 3 cases of CSF of medulloblastoma (M)confirmed by postoperative pathology,then pooled by equal amount as M group;3 cases of CSF of ependymoma (E)confirmed by postoperative pathology,then pooled by e-qual amount as E group;3 cases 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 Cy5 and then pooled.2D-DIGE of labeled samples were run.2D-DIGE gels scanned with 488 nm,532 nm 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 1566 protein spots on gel 1,gel 2 and gel 3 respectively.Biological variation analysis (BVA)module of DeCyder indicated that when average ratio >2.0 or <-2.0,there were differentially expressed pro-tein spots 36 between M and Con,71 between E and Con,52 between M and E.Moreover,there were 8 up-regulated spots only in M group and 10 up-regulated spots only in M group.Conclusion The method of 2D-DIGE has been estab-lished succesfully,which provide a foundation for searching of the protein marker in the CSF of medulloblastoma or ependymoma.