应用定量蛋白质组学方法筛选肺鳞癌转移相关蛋白
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摘要
目的应用激光捕获显微切割(laser capture microdissection,LCM)和荧光差异显示凝胶电泳(differential in-gel electrophoresis,DIGE)技术建立有转移与无转移的人肺鳞癌组织的双向凝胶电泳图谱,筛选并鉴定出有转移和无转移的肺鳞癌组织间差异表达的蛋白质,DeCyder 2D图像分析软件识别差异蛋白质点,质谱鉴定差异表达蛋白质,为研究肺鳞癌转移机制、筛选肺鳞癌转移相关标志物奠定基础。
方法收集术后新鲜肺鳞癌标本,根据病理诊断和临床检查将标本分为2组:有转移的肺鳞癌组(16例)和无转移的肺鳞癌组(12例)。用激光捕获显微切割方法纯化癌细胞,提取细胞总蛋白,采用DIGE技术得到Cy2、Cy3及Cy5荧光素标记的有转移和无转移的肺鳞癌组织凝胶图谱。采用DeCyder 2D图像分析软件进行分析,识别两组间差异表达的蛋白质点。选取差异蛋白质点,胶内酶解后行肽质量指纹分析及网上数据库查询,鉴定差异蛋白质。然后采用Western-Blot和免疫组化验证,膜联蛋白Ⅱ(AnnexinⅡ)和组织蛋白酶D(Cathepsin D)在两组肺鳞癌组织中的表达水平。
结果建立了LCM纯化的有转移与无转移的肺鳞癌组织荧光差异显示凝胶电泳图谱,共识别了24个差异表达蛋白质点,质谱鉴定了14个蛋白质。其中10个蛋白质在有转移的肺鳞癌组织中表达上调,4个蛋白质在有转移肺鳞癌组织中表达下调。蛋白质AnnexinⅡ,Cathepsin D经Western blot和免疫组化检测,在有转移的肺鳞癌组织中表达均高于无转移肺鳞癌组织,与比较蛋白质组学研究结果一致。
结论本实验首次应用LCM结合DIGE技术成功建立了有转移和无转移的肺鳞癌组织间荧光差异表达双向凝胶图谱,鉴定了14个肺鳞癌转移相关蛋白质,并对其中2个差异表达蛋白质进行了验证。这些蛋白质可能在肺鳞癌转移中起到促进或者抑制的作用,为研究肺鳞癌转移机制、筛选肺鳞癌转移相关标志物奠定基础。
Objective The study aims to establish two-dimensional gel electrophoresis(2-DE)map among lung squamous carcinoma(with metastasis/without metastasis)using differential in-gel electrophoresis technique(DIGE),coupled with laser capture microdissection(LCM). The differentially expressed proteins were detected by DeCyder~(TM)2D 6.5 software and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).These data may be useful for better understanding of lung squamous carcinoma metastatic mechanism and uncovering the metastasis-associated biomarkers.
Method In sample preparation,the fresh carcinoma tissue was cut off from the specimen immediately after the specimen was resected and then preserverd in the -80℃refrigeratory.The tissues were classified into 2 groups:lung squamous carcinoma with metastasis and lung squamous carcinoma without metastasis.The former has 16 specimens and the latter has 12 specimens.LCM was utilized to harvest cancer cells from these two groups.Proteins collected from LCM were examined by 2-DE after labeled with CyDye DIGE Fluors Cy2,Cy3 and Cy5.Then the labeled proteins in each gel were visualized using a Typhoon 9000 fluorescence scanner for Cy2,Cy3 and Cy5 dyes.Images were analyzed with the DeCyder~(TM)2D 6.5 software.Protein spots with intensity changes detected by statistical significance were identified by MALDI-TOF-MS.The differentially expressed protein-spots were incised from the gels and digested by trypsin.We acquired the peptide mass fingerprintings(PMF) by MALDI-TOF-MS and identified the proteins by data searching in the Mascot-database.Part of differentially expressed proteins including AnnexinⅡand Cathepsin D were validated by Western blot and immunohistochemistry.
Results Comparative fluorescence differential in-gel electrophoresis map was well established with pure cancer cells of lung squamous carcinoma with metastasis and without metastasis collected by LCM. There were 24 proteins spots differentially expressed between metastasis group and no-metastasis group.All of these proteins spots were selected for mass spectrum and bioinformation analysis.14 proteins were identified.10 of them were upregulated in the group with metastasis,and 4 of them were upregulated in the group without metastasis.AnnexinⅡand Cathepsin D expression were confirmed by Western blot and immunohistochemical analysis.The result showed that these two proteins expression were stronger in lung squmaous tissues with metastasis than lung squmaous tissues without metastasis.The result were in accordance with 2-DE data.
Conclusions We successfully established fluorescence differential in-gel electrophoresis map of lung squamous carcinoma with metastasis and without metastasis by LCM and DIGE technique.14 proteins associated with lung squamous carcinoma metastasis were identified by MALDI-TOF-MS.2 of these proteins were selected to verify the liability of differential proteins.These proteins may accelerate or restrain the progression of lung squamous carcinoma.The data paves the way for better understanding the lung squamous carcinoma metastatic mechanism and identifying potential metastasis-associated markers for lung squamous carcinoma.
方法收集术后新鲜肺鳞癌标本,根据病理诊断和临床检查将标本分为2组:有转移的肺鳞癌组(16例)和无转移的肺鳞癌组(12例)。用激光捕获显微切割方法纯化癌细胞,提取细胞总蛋白,采用DIGE技术得到Cy2、Cy3及Cy5荧光素标记的有转移和无转移的肺鳞癌组织凝胶图谱。采用DeCyder 2D图像分析软件进行分析,识别两组间差异表达的蛋白质点。选取差异蛋白质点,胶内酶解后行肽质量指纹分析及网上数据库查询,鉴定差异蛋白质。然后采用Western-Blot和免疫组化验证,膜联蛋白Ⅱ(AnnexinⅡ)和组织蛋白酶D(Cathepsin D)在两组肺鳞癌组织中的表达水平。
结果建立了LCM纯化的有转移与无转移的肺鳞癌组织荧光差异显示凝胶电泳图谱,共识别了24个差异表达蛋白质点,质谱鉴定了14个蛋白质。其中10个蛋白质在有转移的肺鳞癌组织中表达上调,4个蛋白质在有转移肺鳞癌组织中表达下调。蛋白质AnnexinⅡ,Cathepsin D经Western blot和免疫组化检测,在有转移的肺鳞癌组织中表达均高于无转移肺鳞癌组织,与比较蛋白质组学研究结果一致。
结论本实验首次应用LCM结合DIGE技术成功建立了有转移和无转移的肺鳞癌组织间荧光差异表达双向凝胶图谱,鉴定了14个肺鳞癌转移相关蛋白质,并对其中2个差异表达蛋白质进行了验证。这些蛋白质可能在肺鳞癌转移中起到促进或者抑制的作用,为研究肺鳞癌转移机制、筛选肺鳞癌转移相关标志物奠定基础。
Objective The study aims to establish two-dimensional gel electrophoresis(2-DE)map among lung squamous carcinoma(with metastasis/without metastasis)using differential in-gel electrophoresis technique(DIGE),coupled with laser capture microdissection(LCM). The differentially expressed proteins were detected by DeCyder~(TM)2D 6.5 software and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).These data may be useful for better understanding of lung squamous carcinoma metastatic mechanism and uncovering the metastasis-associated biomarkers.
Method In sample preparation,the fresh carcinoma tissue was cut off from the specimen immediately after the specimen was resected and then preserverd in the -80℃refrigeratory.The tissues were classified into 2 groups:lung squamous carcinoma with metastasis and lung squamous carcinoma without metastasis.The former has 16 specimens and the latter has 12 specimens.LCM was utilized to harvest cancer cells from these two groups.Proteins collected from LCM were examined by 2-DE after labeled with CyDye DIGE Fluors Cy2,Cy3 and Cy5.Then the labeled proteins in each gel were visualized using a Typhoon 9000 fluorescence scanner for Cy2,Cy3 and Cy5 dyes.Images were analyzed with the DeCyder~(TM)2D 6.5 software.Protein spots with intensity changes detected by statistical significance were identified by MALDI-TOF-MS.The differentially expressed protein-spots were incised from the gels and digested by trypsin.We acquired the peptide mass fingerprintings(PMF) by MALDI-TOF-MS and identified the proteins by data searching in the Mascot-database.Part of differentially expressed proteins including AnnexinⅡand Cathepsin D were validated by Western blot and immunohistochemistry.
Results Comparative fluorescence differential in-gel electrophoresis map was well established with pure cancer cells of lung squamous carcinoma with metastasis and without metastasis collected by LCM. There were 24 proteins spots differentially expressed between metastasis group and no-metastasis group.All of these proteins spots were selected for mass spectrum and bioinformation analysis.14 proteins were identified.10 of them were upregulated in the group with metastasis,and 4 of them were upregulated in the group without metastasis.AnnexinⅡand Cathepsin D expression were confirmed by Western blot and immunohistochemical analysis.The result showed that these two proteins expression were stronger in lung squmaous tissues with metastasis than lung squmaous tissues without metastasis.The result were in accordance with 2-DE data.
Conclusions We successfully established fluorescence differential in-gel electrophoresis map of lung squamous carcinoma with metastasis and without metastasis by LCM and DIGE technique.14 proteins associated with lung squamous carcinoma metastasis were identified by MALDI-TOF-MS.2 of these proteins were selected to verify the liability of differential proteins.These proteins may accelerate or restrain the progression of lung squamous carcinoma.The data paves the way for better understanding the lung squamous carcinoma metastatic mechanism and identifying potential metastasis-associated markers for lung squamous carcinoma.
引文
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