髓母细胞瘤与室管膜瘤患者脑脊液比较蛋白质组学研究
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摘要
背景：髓母细胞瘤与室管膜瘤都为恶性神经上皮肿瘤,髓母细胞瘤较室管膜瘤恶性程度更高,皆好发于儿童,在所有儿童中枢神经系统肿瘤的发病率中分别位居第二、第三,对儿童健康影响极大。由于髓母细胞瘤和室管膜瘤是四脑室内最为常见的肿瘤,与脑干关系密切,手术不易彻底切除,加之两种肿瘤皆具有沿脑脊液循环途径种植转移的倾向,导致其治愈率低,预后差。脑脊液因与这两种肿瘤的接近性和临床可行性而具有重要的临床诊断价值。因此在脑脊液中寻找到肿瘤生物标志物,将有助于早期诊断、鉴别诊断,判断预后,监测肿瘤复发及治疗反应。
蛋白质组学是采用高分辨率的蛋白质分离手段结合高通量的蛋白质鉴定技术,全景式地研究在特定时间和空间上的蛋白质表达谱。而比较蛋白质组学研究差异样品间蛋白质表达的变化。比较蛋白质组学包括两大主要技术,即蛋白质分离技术与蛋白质鉴定技术。双向荧光差异凝胶电泳(2D-DIGE)是先进的蛋白质分离技术,能显著提高微量蛋白检测的灵敏度,而且因其引入内标概念,更有利于对差异表达蛋白质的准确检测。质谱技术为蛋白质组学研究中主要的蛋白质鉴定技术,包括一级质谱(MS)和二级质谱(MS/MS).二级质谱又称串联质谱,可以更精确、更灵敏地分析、鉴定蛋白质样品。
目的：本研究应用比较蛋白质组学技术,对髓母细胞瘤脑脊液、室管膜瘤脑脊液及正常对照组脑脊液进行比较蛋白质组学研究,分离、鉴定差异表达蛋白质,以期为髓母细胞瘤、室管膜瘤脑脊液生物标志物的探寻提供线索、为揭示髓母细胞瘤及室管膜瘤发病机制奠定理论及实验基础。同时结合基因芯片技术,建立髓母细胞瘤、室管膜瘤和小脑组织的基因表达谱,以明确脑脊液中差异蛋白质的编码基因在相应肿瘤组织中的表达情况,为脑脊液中差异蛋白质来源推测提供一定的实验依据。
方法：
实验分为三组：髓母细胞瘤组：选取9例髓母细胞瘤患者脑脊液、3例髓母细胞瘤肿瘤组织；室管膜瘤组：9例室管膜瘤患者脑脊液、3例室管膜瘤肿瘤组织；正常对照组：6例无其他神经系统病变的头痛患者的脑脊液(脑脊液生化检查正常),3例外伤手术减压的小脑组织。
(1)三组之间脑脊液比较蛋白质组学定量分析：①使用10%浓度的TC/丙酮分别沉淀三组脑脊液蛋白,Bradford法定量蛋白。②分析胶双向电泳：每组样品各取50μg进行荧光标记,采用CyDye最小标记法标记蛋白,Cy2标记内标组(三组蛋白质等量混合为内标组),Cy3、Cy5标记实验组或对照组,标记蛋白混合后(150μg)进行2D-DIGE分离蛋白质。第一向等电聚焦电泳(IEF)：使用24cm长的固相pH梯度(IPG)干胶条(pH3～10)进行等电聚焦；第二向SDS-PAGE电泳：IPG胶条经过2次平衡后,转移至12%SDS-PAGE凝胶中进行电泳。用激光扫描仪于荧光模式下进行Cy2、Cy3、Cy5成像扫描,Cy2、Cy3、Cy5标记的蛋白成像分别由488nm、532nm、633n的激发波长收集,蛋白点分别显示为蓝色、绿色和红色。使用DeCyder图像分析软件中的胶内差异分析(DIA)模块和生物学差异分析(BVA)模块分析图像结果,确定差异蛋白点。③制备胶双向电泳：从两个实验组和1个对照组中各取600μg蛋白进行双向凝胶电泳,考马斯亮兰染色。④利用基质辅助激光解吸附电离-飞行时间串联质谱(MALDI-TOF-TOF)选择性鉴定差异显著的蛋白质,主要为在髓母细胞瘤和/或室管膜瘤脑脊液中表达上调2倍以上的蛋白质。将考染的制备胶与分析胶进行比较,切取制备胶上与分析胶上差异表达点相匹配的蛋白斑点,使用胰蛋白酶进行胶内酶解蛋白,由MALDI-TOF/TOF质谱仪获得一级和二级肽指纹图谱,利用GPS软件在人IPI数据库中检索,采用MS& MS/MS联合搜索的方式,将蛋白质打分大于60分、置信水平在95%以上作为数据取舍标准。
(2)三组之间组织比较基因表达谱定量分析：建立髓母细胞瘤、室管膜瘤、小脑组织基因差异表达数据库。①分别提取髓母细胞瘤、室管膜瘤组织及小脑组织总RNA,并定量、质检和纯化总RNA；②cDNA的合成和纯化；③体外转录(IVT)合成生物素标记的cRNA;④cRNA片段化后与Affymetrix Human Genome U133plus 2.0基因表达谱芯片杂交。⑤洗脱、扫描芯片,应用GCOS软件进行数据分析。
结果：
(1)脑脊液2D-DIGE结果显示：
①髓母细胞瘤组与正常对照组差异表达在2倍以上的蛋白质点共35个,其中17个蛋白质点表达上调,18个蛋白质点表达下调。
②室管膜瘤组与正常对照组差异表达在2倍以上的蛋白质点共74个,其中38个蛋白质点表达上调,36个蛋白质点表达下调。
③髓母细胞瘤组与室管膜瘤组差异表达在2倍以上的蛋白质点共59个,其中12个蛋白质点表达上调,47个蛋白质点表达下调。
(2)质谱鉴定结果显示：选择性对28个上调的蛋白质点进行MALDI-TOF/TOF鉴定,其中18个蛋白质点成功鉴定,对应12个蛋白质。
①补体因子H异构体1、α-2巨球蛋白、血清转铁蛋白、纤维蛋白原β链、免疫球蛋白重链恒定区α1、触珠蛋白异构体2前蛋白原、触珠蛋白(高度类似)、β肌动蛋白(高度类似)在室管膜瘤组中较正常对照组和髓母细胞瘤组显著升高,而在髓母细胞瘤组与正常对照组之间无显著差异。
②补体因子B异构体1(片段)在室管膜瘤组和髓母细胞瘤组中较正常对照组皆显著增高,而在两种肿瘤组之间表达无显著差异。
③α-1抗胰蛋白酶异构体1和神经细胞粘附分子异构体3在髓母细胞瘤组中较正常对照组和室管膜瘤组中显著升高,而室管膜瘤组与正常对照组之间无显著差异。
④未知功能蛋白ALB和未知功能蛋白DKFZp686M08189仅在髓母细胞瘤组中表达升高。
(3)组织基因表达谱结果显示：
①补体因子H编码基因仅在髓母细胞瘤中探测到表达而触珠蛋白编码基因仅在室管膜瘤中探测到表达。
②α-1抗胰蛋白酶编码基因在小脑组织、室管膜瘤组织探测到表达,而在髓母细胞瘤中未探测到表达。
③α-2巨球蛋白编码基因、神经细胞粘附分子编码基因、血清转铁蛋白编码基因和β肌动蛋白编码基因在髓母细胞瘤、室管膜瘤、小脑组织中都探测到表达。
④补体因子B编码基因、纤维蛋白原β链编码基因和免疫球蛋白重链恒定区α1编码基因在髓母细胞瘤、室管膜瘤、小脑组织中都未探测到表达。
结论：髓母细胞瘤脑脊液、室管膜瘤脑脊液、正常对照脑脊液2D-DIGE图谱之间存在差异,选择性鉴定出12个在髓母细胞瘤脑脊液和/或室管膜瘤脑脊液中显著增高的蛋白质,为在脑脊液中探寻两种肿瘤的潜在生物标志物提供了有意义的研究线索；基因表达谱研究有助于推测脑脊液中差异蛋白质来源,为选取价值较大的差异蛋白质进行深入研究指出方向。
Background Both medulloblastoma and ependymoma are neuroepithelial tumors. Medulloblastoma has a higher degree of malignancy than ependymoma. The incidence of medulloblastoma ranked second in central nervous system tumors in childhood, while the incidence of ependymoma ranked third. They have a great bad impact on children's health. Because they are the most common tumors in the fourth ventricle and get close to brain stem, it is difficult to resect the tumors completely. Moreover, they have a tendency to spread through the cerebrospinal fluid (CSF), which results in the low rate of cure with poor prognosis. CSF has the important clinical value because of contact with the tumors and the clinical feasibility of the approach. Therefore, if the tumor biomarkers are found in the CSF, it will be helpful to make the early diagnosis as well as differential diagnosis, to determine prognosis, to monitor tumor recurrence and to observe treatment response.
Proteomics study the protein expression profiles in the certain time and surroundings by means of the combination of high-resolution protein separation technique and high-throughput protein identification technique. Comparative proteomics analyze the changes of protein expression level between different samples. Comparative proteomics contain two main kinds of technique, protein separation technique and protein identification technique. Two-dimensional differential in-gel electrophoresis (2D-DIGE), an advanced protein separation technique, can significantly improve the sensitivity of the detection of trace protein. Introducing the concept of internal standard,2D-DIGE is more conducive to detect the differentially expressed proteins accurately. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) in proteomic research are the major protein identification techniques. MS/MS is more accurate and more sensitive for identification of protein.Objective To provide the clues in search of biomarkers in CSF of medulloblastoma and ependymoma and to offer theoretical and experimental basis for revealing the pathogenesis of medulloblastoma and ependymoma, the differentially expressed proteins in CSF between medulloblastoma, ependymoma and normal control group are isolated and identified via the comparative proteomic techniques. Combined with genechip technology, gene expression profiles of medulloblastoma, ependymoma and cerebellar tissue are established in order to identify differences in the mRNA expression of the genes encoding the differentially expressed proteins. The study of gene expression profile provides some experimental evidences to speculate the sources of differentially expressed proteins in CSF.
Methods
The experiment was divided three groups:medulloblastoma group:nine cases of CSF in patients with medulloblastoma, three cases of medulloblastoma tissue; ependymoma group: nine cases of CSF in patients with ependymoma, three cases of ependymoma tissue; normal control group:six cases of CSF in patients with headache who had no other neurological disorders(normal CSF examination),3 cases of cerebellum tissue from traumatic surgical decompression.
(1) Quantitative analysis of comparative proteomics between the three groups:①With 10% TCA/acetone, total proteins in CSF of three groups were precipitated and extracted respectively. Then the proteins were quantified with Bradford method.②Analytical gel electrophoresis:2D-DIGE was used. The internal standard comprised equal amounts of proteins extracted from three groups. Internal standard and proteins from two of three groups were minimally labeled with fluorescent dyes, Cy2、Cy3 and Cy5, then the three samples pooled. Every sample's labelled protein is 50μg. First dimension IEF was performed with 24 cm immobilized pH 3-10 linear gradient dry strip for isoelectric focusing. Then, the proteins were isolated according to the difference of isoelectric point. Second dimension SDS-PAGE was performed with homogeneous 12.5% gels after equilibration twice. Then, the proteins were isolated according to the difference of molecular weight. The fluorescence signals of the three differently CyDye-labeled protein samples were imaged by using a laser scanner and wavelengths of 488 nm (Cy2, blue),532 nm (Cy3, green) and 633 nm (Cy5, red). The detection and quantification of protein spots were performed for the differential in-gel analysis (DIA) module of DeCyder software. Biological variation analysis (BVA) module of DeCyder can provide data on differential protein expression levels beteeen the three groups.③Preparative gel electrophoresis:600μg proteins from every group run 2D-PAGE respectively. Then the gels were stained with Coomassie brilliant blue.④Protein identification:differentially expressed proteins were selectively identified by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS), Mainly up-regulated proteins in 2 fold in CSF of medulloblastoma and/or ependymoma. After comparison between preparative gel and analytical gel, the spots in preparative gel that matched differentially expressed spots in analytical gel were excised. After enzymolysis of trypsin, MS and MS/MS were obtained with mass spectrometer. Peptide mass data were searched in data IPI human with GPS software.
(2) Quantitative analysis of comparative gene expression profiles between the three groups:①Total RNA of medulloblastoma, ependymoma and cerebellum were extracted respectively. Then quantification, clean up and checking of total RNA were performed.②Synthesis and clean up of cDNA.③Synthesis of biotin-labeled cRNA in vitro transcription.④Fragmenting the cRNA and hybridization with Affymetrix Human Genome U133plus 2.0 genechip.⑤Elution and scan of the genechip.⑥Data analysis with GCOS software.
Results
(1) 2D-DIGE analysis showed that the levels of 35 protein spots were significantly altered in medulloblastoma CSF compared with control CSF when average ratio of the matched spot was assigned as>2.0 or<-2.0, of which 17 spots were up-regulated and 18 spots were down-regulated.
(2) 2D-DIGE analysis revealed that levels of 74 protein spots were significantly changed in ependymoma CSF compared with control CSF when average ratio>2.0 or<-2.0, of which 38 spots were up-regulated and 36 spots were down-regulated.
(3) 2D-DIGE analysis demonstrated that levels of 59 protein spots were significantly altered in medulloblastoma CSF compared with ependymoma CSF when average ratio>2.0 or<-2.0, of which 12 spots were up-regulated and 47 spots were down-regulated.
(4) 28 up-regulated (average ratio>2.0) spots were selected to be identified with MALDI-TOF/TOF, of which 18 spots were identified successfully, corresponding to 12 unique proteins.
(5) Isoform 1 of complement factor H, alpha-2-macroglobulin, serotransferrin, fibrinogen beta chain, immunoglobulin heavy constant alpha 1, haptoglobin isoform 2 preproprotein, highly similar to Haptoglobin and highly similar to actin cytoplasmic 1 (beta-actin) were markedly increased in ependymoma CSF compared with the medulloblastoma CSF and control CSF, which were not significantly altered between the medulloblastoma CSF and control CSF. Isoform 1 of complement factor B was markedly increased in medulloblastoma CSF and ependymoma CSF compared with control CSF, which was not significantly changed between ependymoma CSF and the medulloblastoma CSF. Isoform 1 of alpha-1-antitrypsin and isoform 3 of neuronal cell adhesion molecule was markedly increased in medulloblastoma CSF compared with ependymoma CSF and control CSF, which was not significantly altered between ependymoma CSF and control CSF. Putative uncharacterized protein ALB and putative uncharacterized protein DKFZp686M08189 were markedly increased only in medulloblastoma CSF.
(6) Gene expression profiles:the gene encoding complement factor H was detected to express only in medulloblastoma, while the gene encoding haptoglobin was detected to express only in ependymoma. The gene encoding alpha-1-antitrypsin was detected to express in ependymoma and cerebellum, but not in medulloblastoma. The gene encoding alpha-2-macroglobulin, the gene encoding neuronal cell adhesion molecule, the gene encoding serotransferrin and the gene encoding beta-actin was detected to express in medulloblastoma, ependymoma and cerebellum. The gene encoding complement factor B, the gene encoding fibrinogen beta chain and the gene encoding immunoglobulin heavy constant alpha 1 were not detected to express in medulloblastoma, ependymoma and cerebellum.
Conclusions There were some significantly differential protein spots in 2D-DIGE of CSF between medulloblastoma, ependymoma and cerebellum.12 differentially expressed proteins in CSF of medulloblastoma and/or ependymoma were identified, which provided meaningful clues for exploring the potential biomarkers of two kinds of tumor in CSF. However, these potential biomarkers needed further validation. The study of gene expression profile was helpful to speculate the the sources of differentially expressed proteins in CSF and pointed out the direction to select the valuable proteins for further study.
蛋白质组学是采用高分辨率的蛋白质分离手段结合高通量的蛋白质鉴定技术,全景式地研究在特定时间和空间上的蛋白质表达谱。而比较蛋白质组学研究差异样品间蛋白质表达的变化。比较蛋白质组学包括两大主要技术,即蛋白质分离技术与蛋白质鉴定技术。双向荧光差异凝胶电泳(2D-DIGE)是先进的蛋白质分离技术,能显著提高微量蛋白检测的灵敏度,而且因其引入内标概念,更有利于对差异表达蛋白质的准确检测。质谱技术为蛋白质组学研究中主要的蛋白质鉴定技术,包括一级质谱(MS)和二级质谱(MS/MS).二级质谱又称串联质谱,可以更精确、更灵敏地分析、鉴定蛋白质样品。
目的：本研究应用比较蛋白质组学技术,对髓母细胞瘤脑脊液、室管膜瘤脑脊液及正常对照组脑脊液进行比较蛋白质组学研究,分离、鉴定差异表达蛋白质,以期为髓母细胞瘤、室管膜瘤脑脊液生物标志物的探寻提供线索、为揭示髓母细胞瘤及室管膜瘤发病机制奠定理论及实验基础。同时结合基因芯片技术,建立髓母细胞瘤、室管膜瘤和小脑组织的基因表达谱,以明确脑脊液中差异蛋白质的编码基因在相应肿瘤组织中的表达情况,为脑脊液中差异蛋白质来源推测提供一定的实验依据。
方法：
实验分为三组：髓母细胞瘤组：选取9例髓母细胞瘤患者脑脊液、3例髓母细胞瘤肿瘤组织；室管膜瘤组：9例室管膜瘤患者脑脊液、3例室管膜瘤肿瘤组织；正常对照组：6例无其他神经系统病变的头痛患者的脑脊液(脑脊液生化检查正常),3例外伤手术减压的小脑组织。
(1)三组之间脑脊液比较蛋白质组学定量分析：①使用10%浓度的TC/丙酮分别沉淀三组脑脊液蛋白,Bradford法定量蛋白。②分析胶双向电泳：每组样品各取50μg进行荧光标记,采用CyDye最小标记法标记蛋白,Cy2标记内标组(三组蛋白质等量混合为内标组),Cy3、Cy5标记实验组或对照组,标记蛋白混合后(150μg)进行2D-DIGE分离蛋白质。第一向等电聚焦电泳(IEF)：使用24cm长的固相pH梯度(IPG)干胶条(pH3～10)进行等电聚焦；第二向SDS-PAGE电泳：IPG胶条经过2次平衡后,转移至12%SDS-PAGE凝胶中进行电泳。用激光扫描仪于荧光模式下进行Cy2、Cy3、Cy5成像扫描,Cy2、Cy3、Cy5标记的蛋白成像分别由488nm、532nm、633n的激发波长收集,蛋白点分别显示为蓝色、绿色和红色。使用DeCyder图像分析软件中的胶内差异分析(DIA)模块和生物学差异分析(BVA)模块分析图像结果,确定差异蛋白点。③制备胶双向电泳：从两个实验组和1个对照组中各取600μg蛋白进行双向凝胶电泳,考马斯亮兰染色。④利用基质辅助激光解吸附电离-飞行时间串联质谱(MALDI-TOF-TOF)选择性鉴定差异显著的蛋白质,主要为在髓母细胞瘤和/或室管膜瘤脑脊液中表达上调2倍以上的蛋白质。将考染的制备胶与分析胶进行比较,切取制备胶上与分析胶上差异表达点相匹配的蛋白斑点,使用胰蛋白酶进行胶内酶解蛋白,由MALDI-TOF/TOF质谱仪获得一级和二级肽指纹图谱,利用GPS软件在人IPI数据库中检索,采用MS& MS/MS联合搜索的方式,将蛋白质打分大于60分、置信水平在95%以上作为数据取舍标准。
(2)三组之间组织比较基因表达谱定量分析：建立髓母细胞瘤、室管膜瘤、小脑组织基因差异表达数据库。①分别提取髓母细胞瘤、室管膜瘤组织及小脑组织总RNA,并定量、质检和纯化总RNA；②cDNA的合成和纯化；③体外转录(IVT)合成生物素标记的cRNA;④cRNA片段化后与Affymetrix Human Genome U133plus 2.0基因表达谱芯片杂交。⑤洗脱、扫描芯片,应用GCOS软件进行数据分析。
结果：
(1)脑脊液2D-DIGE结果显示：
①髓母细胞瘤组与正常对照组差异表达在2倍以上的蛋白质点共35个,其中17个蛋白质点表达上调,18个蛋白质点表达下调。
②室管膜瘤组与正常对照组差异表达在2倍以上的蛋白质点共74个,其中38个蛋白质点表达上调,36个蛋白质点表达下调。
③髓母细胞瘤组与室管膜瘤组差异表达在2倍以上的蛋白质点共59个,其中12个蛋白质点表达上调,47个蛋白质点表达下调。
(2)质谱鉴定结果显示：选择性对28个上调的蛋白质点进行MALDI-TOF/TOF鉴定,其中18个蛋白质点成功鉴定,对应12个蛋白质。
①补体因子H异构体1、α-2巨球蛋白、血清转铁蛋白、纤维蛋白原β链、免疫球蛋白重链恒定区α1、触珠蛋白异构体2前蛋白原、触珠蛋白(高度类似)、β肌动蛋白(高度类似)在室管膜瘤组中较正常对照组和髓母细胞瘤组显著升高,而在髓母细胞瘤组与正常对照组之间无显著差异。
②补体因子B异构体1(片段)在室管膜瘤组和髓母细胞瘤组中较正常对照组皆显著增高,而在两种肿瘤组之间表达无显著差异。
③α-1抗胰蛋白酶异构体1和神经细胞粘附分子异构体3在髓母细胞瘤组中较正常对照组和室管膜瘤组中显著升高,而室管膜瘤组与正常对照组之间无显著差异。
④未知功能蛋白ALB和未知功能蛋白DKFZp686M08189仅在髓母细胞瘤组中表达升高。
(3)组织基因表达谱结果显示：
①补体因子H编码基因仅在髓母细胞瘤中探测到表达而触珠蛋白编码基因仅在室管膜瘤中探测到表达。
②α-1抗胰蛋白酶编码基因在小脑组织、室管膜瘤组织探测到表达,而在髓母细胞瘤中未探测到表达。
③α-2巨球蛋白编码基因、神经细胞粘附分子编码基因、血清转铁蛋白编码基因和β肌动蛋白编码基因在髓母细胞瘤、室管膜瘤、小脑组织中都探测到表达。
④补体因子B编码基因、纤维蛋白原β链编码基因和免疫球蛋白重链恒定区α1编码基因在髓母细胞瘤、室管膜瘤、小脑组织中都未探测到表达。
结论：髓母细胞瘤脑脊液、室管膜瘤脑脊液、正常对照脑脊液2D-DIGE图谱之间存在差异,选择性鉴定出12个在髓母细胞瘤脑脊液和/或室管膜瘤脑脊液中显著增高的蛋白质,为在脑脊液中探寻两种肿瘤的潜在生物标志物提供了有意义的研究线索；基因表达谱研究有助于推测脑脊液中差异蛋白质来源,为选取价值较大的差异蛋白质进行深入研究指出方向。
Background Both medulloblastoma and ependymoma are neuroepithelial tumors. Medulloblastoma has a higher degree of malignancy than ependymoma. The incidence of medulloblastoma ranked second in central nervous system tumors in childhood, while the incidence of ependymoma ranked third. They have a great bad impact on children's health. Because they are the most common tumors in the fourth ventricle and get close to brain stem, it is difficult to resect the tumors completely. Moreover, they have a tendency to spread through the cerebrospinal fluid (CSF), which results in the low rate of cure with poor prognosis. CSF has the important clinical value because of contact with the tumors and the clinical feasibility of the approach. Therefore, if the tumor biomarkers are found in the CSF, it will be helpful to make the early diagnosis as well as differential diagnosis, to determine prognosis, to monitor tumor recurrence and to observe treatment response.
Proteomics study the protein expression profiles in the certain time and surroundings by means of the combination of high-resolution protein separation technique and high-throughput protein identification technique. Comparative proteomics analyze the changes of protein expression level between different samples. Comparative proteomics contain two main kinds of technique, protein separation technique and protein identification technique. Two-dimensional differential in-gel electrophoresis (2D-DIGE), an advanced protein separation technique, can significantly improve the sensitivity of the detection of trace protein. Introducing the concept of internal standard,2D-DIGE is more conducive to detect the differentially expressed proteins accurately. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) in proteomic research are the major protein identification techniques. MS/MS is more accurate and more sensitive for identification of protein.Objective To provide the clues in search of biomarkers in CSF of medulloblastoma and ependymoma and to offer theoretical and experimental basis for revealing the pathogenesis of medulloblastoma and ependymoma, the differentially expressed proteins in CSF between medulloblastoma, ependymoma and normal control group are isolated and identified via the comparative proteomic techniques. Combined with genechip technology, gene expression profiles of medulloblastoma, ependymoma and cerebellar tissue are established in order to identify differences in the mRNA expression of the genes encoding the differentially expressed proteins. The study of gene expression profile provides some experimental evidences to speculate the sources of differentially expressed proteins in CSF.
Methods
The experiment was divided three groups:medulloblastoma group:nine cases of CSF in patients with medulloblastoma, three cases of medulloblastoma tissue; ependymoma group: nine cases of CSF in patients with ependymoma, three cases of ependymoma tissue; normal control group:six cases of CSF in patients with headache who had no other neurological disorders(normal CSF examination),3 cases of cerebellum tissue from traumatic surgical decompression.
(1) Quantitative analysis of comparative proteomics between the three groups:①With 10% TCA/acetone, total proteins in CSF of three groups were precipitated and extracted respectively. Then the proteins were quantified with Bradford method.②Analytical gel electrophoresis:2D-DIGE was used. The internal standard comprised equal amounts of proteins extracted from three groups. Internal standard and proteins from two of three groups were minimally labeled with fluorescent dyes, Cy2、Cy3 and Cy5, then the three samples pooled. Every sample's labelled protein is 50μg. First dimension IEF was performed with 24 cm immobilized pH 3-10 linear gradient dry strip for isoelectric focusing. Then, the proteins were isolated according to the difference of isoelectric point. Second dimension SDS-PAGE was performed with homogeneous 12.5% gels after equilibration twice. Then, the proteins were isolated according to the difference of molecular weight. The fluorescence signals of the three differently CyDye-labeled protein samples were imaged by using a laser scanner and wavelengths of 488 nm (Cy2, blue),532 nm (Cy3, green) and 633 nm (Cy5, red). The detection and quantification of protein spots were performed for the differential in-gel analysis (DIA) module of DeCyder software. Biological variation analysis (BVA) module of DeCyder can provide data on differential protein expression levels beteeen the three groups.③Preparative gel electrophoresis:600μg proteins from every group run 2D-PAGE respectively. Then the gels were stained with Coomassie brilliant blue.④Protein identification:differentially expressed proteins were selectively identified by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS), Mainly up-regulated proteins in 2 fold in CSF of medulloblastoma and/or ependymoma. After comparison between preparative gel and analytical gel, the spots in preparative gel that matched differentially expressed spots in analytical gel were excised. After enzymolysis of trypsin, MS and MS/MS were obtained with mass spectrometer. Peptide mass data were searched in data IPI human with GPS software.
(2) Quantitative analysis of comparative gene expression profiles between the three groups:①Total RNA of medulloblastoma, ependymoma and cerebellum were extracted respectively. Then quantification, clean up and checking of total RNA were performed.②Synthesis and clean up of cDNA.③Synthesis of biotin-labeled cRNA in vitro transcription.④Fragmenting the cRNA and hybridization with Affymetrix Human Genome U133plus 2.0 genechip.⑤Elution and scan of the genechip.⑥Data analysis with GCOS software.
Results
(1) 2D-DIGE analysis showed that the levels of 35 protein spots were significantly altered in medulloblastoma CSF compared with control CSF when average ratio of the matched spot was assigned as>2.0 or<-2.0, of which 17 spots were up-regulated and 18 spots were down-regulated.
(2) 2D-DIGE analysis revealed that levels of 74 protein spots were significantly changed in ependymoma CSF compared with control CSF when average ratio>2.0 or<-2.0, of which 38 spots were up-regulated and 36 spots were down-regulated.
(3) 2D-DIGE analysis demonstrated that levels of 59 protein spots were significantly altered in medulloblastoma CSF compared with ependymoma CSF when average ratio>2.0 or<-2.0, of which 12 spots were up-regulated and 47 spots were down-regulated.
(4) 28 up-regulated (average ratio>2.0) spots were selected to be identified with MALDI-TOF/TOF, of which 18 spots were identified successfully, corresponding to 12 unique proteins.
(5) Isoform 1 of complement factor H, alpha-2-macroglobulin, serotransferrin, fibrinogen beta chain, immunoglobulin heavy constant alpha 1, haptoglobin isoform 2 preproprotein, highly similar to Haptoglobin and highly similar to actin cytoplasmic 1 (beta-actin) were markedly increased in ependymoma CSF compared with the medulloblastoma CSF and control CSF, which were not significantly altered between the medulloblastoma CSF and control CSF. Isoform 1 of complement factor B was markedly increased in medulloblastoma CSF and ependymoma CSF compared with control CSF, which was not significantly changed between ependymoma CSF and the medulloblastoma CSF. Isoform 1 of alpha-1-antitrypsin and isoform 3 of neuronal cell adhesion molecule was markedly increased in medulloblastoma CSF compared with ependymoma CSF and control CSF, which was not significantly altered between ependymoma CSF and control CSF. Putative uncharacterized protein ALB and putative uncharacterized protein DKFZp686M08189 were markedly increased only in medulloblastoma CSF.
(6) Gene expression profiles:the gene encoding complement factor H was detected to express only in medulloblastoma, while the gene encoding haptoglobin was detected to express only in ependymoma. The gene encoding alpha-1-antitrypsin was detected to express in ependymoma and cerebellum, but not in medulloblastoma. The gene encoding alpha-2-macroglobulin, the gene encoding neuronal cell adhesion molecule, the gene encoding serotransferrin and the gene encoding beta-actin was detected to express in medulloblastoma, ependymoma and cerebellum. The gene encoding complement factor B, the gene encoding fibrinogen beta chain and the gene encoding immunoglobulin heavy constant alpha 1 were not detected to express in medulloblastoma, ependymoma and cerebellum.
Conclusions There were some significantly differential protein spots in 2D-DIGE of CSF between medulloblastoma, ependymoma and cerebellum.12 differentially expressed proteins in CSF of medulloblastoma and/or ependymoma were identified, which provided meaningful clues for exploring the potential biomarkers of two kinds of tumor in CSF. However, these potential biomarkers needed further validation. The study of gene expression profile was helpful to speculate the the sources of differentially expressed proteins in CSF and pointed out the direction to select the valuable proteins for further study.
引文
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