宫颈癌紫杉醇联合顺铂新辅助化疗前后蛋白质组学研究
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摘要
目的：
采用双向凝胶电泳(two-dimensional gelelectrophoresis,2-DE)结合质谱分析技术比较对紫杉醇(paclitaxel, T)联合顺铂(cisplatin, P)新辅助化疗作用敏感的宫颈癌组织化疗前后蛋白质组的差异,建立TP化疗前后的宫颈癌2—DE图谱,识别和鉴定差异表达的蛋白质,初步确定宫颈癌中与TP作用相关的蛋白质,研究其作用机制,为改善宫颈癌患者TP新辅助化疗疗效、研制新的肿瘤靶向药物提供实验及理论依据。
方法：
1、选取6例宫颈癌住院病人,纳入标准为：①化疗前为局部晚期菜花型；②采用紫杉醇加顺铂新辅助化疗：均化疗1次,紫杉醇175mg／m2,顺铂60mg／m2；③化疗10天后评价疗效为显效,肿瘤体积缩小50%以上；④约化疗后15天行广泛性子宫切除术及盆腔淋巴结清扫术；⑤术后病理诊断均为中分化鳞癌。宫颈癌组织化疗前后标本收集后立即-80℃保存。提取组织总蛋白,采用2-DE分离蛋白质,经考马斯亮蓝染色、图像扫描,得到化疗前后的双向电泳图谱,采用PD-quest7.0软件进行匹配和差异分析,识别两组之间表达差异蛋白点。从胶中切取表达差异明显的蛋白点,用MALDI—TOF—TOF—MS方法进行质谱分析,获取质谱图,数据库搜索鉴定蛋白质。
2、根据获得的差异蛋白的分类及抗体购买的难易程度选择4个差异表达蛋白,应用Westem Blot方法进行验证,检测其在以上6例宫颈癌组织标本中化疗前后的表达变化,观察它们在化疗前后的表达是否与蛋白质组学方法检测结果一致。以蛋白质组学所用提取的组织蛋白为研究对象,制备PAGE胶,测蛋白浓度后等量上样,SDS-PAGE电泳,PVDF转膜,脱脂奶粉封闭,抗体孵育,HRP-ECL发光法显色,胶片定影、扫描、分析。以GAPDH为内参。
结果：
1、建立了分辨率高、重复性好的宫颈癌化疗前后的双向凝胶电泳图谱。化疗前蛋白点598士64个,化疗后蛋白点673士79个,组间平均匹配率为85.4%。
2、两组间差异表达蛋白质点数为102个,从中选择表达差异明显的13个点进行质谱分析,全部鉴定成功,其中化疗后与化疗前相比,11个蛋白下调,2个蛋白上调。化疗后表达下调的蛋白：辅肌动蛋白α1(actinin alpha 1)、核纤层蛋白B1 (lamin B1)、真核翻译延长因子1γ(Eukaryotic translation elongation factor 1 gamma)、烯醇化酶1(enolase 1)、膜联蛋白1 (annexinⅠ)、上皮细胞标记蛋白1(epithelial cell marker protein 1)、醛缩酶A(aldolase A)、热休克蛋白27(heat shock protein 27)、丙酮酸激酶(pyruvate kinase)、角蛋白Ⅱ型(cytokeratin typeⅡ)、热休克蛋白70(heat shock protein 70);化疗后表达上调的蛋白：载脂蛋白A1(apolipoprotein A-1)、膜联蛋白V (annexin V)。差异蛋白可归纳为3类：①细胞骨架蛋白：辅肌动蛋白α1,核纤层蛋白B1,角蛋白Ⅱ型,膜联蛋白工,膜联蛋白V；②糖酵解酶类：烯醇化酶1,醛缩酶A,丙酮酸激酶；③其它：真核翻译延长因子1γ,上皮细胞标记蛋白1,热休克蛋白27,热休克蛋白70,载脂蛋白Al。
3、选择HSP70、HSP27、烯醇化酶1、醛缩酶A用Western blot技术进行验证,结果显示这四种蛋白在化疗前组中的表达高于化疗后组。该结果与蛋白质组学研究结果一致。
结论：
1、本研究通过提取宫颈癌化疗前后的总蛋白质,进行双向凝胶电泳,建立了宫颈癌紫杉醇联合顺铂新辅助化疗前后的凝胶电泳图谱,并应用PD—quest7.0软件对获取的凝胶图像进行分析,发现差异表达蛋白质。表明TP能够导致人宫颈癌蛋白质表达谱的改变。
2、本研究应用MALDI—TOF—TOF—MS方法分析部分差异表达明显的蛋白质点,获取肽质量指纹图谱,搜索数据库鉴定出了13个差异蛋白质。这些差异表达的蛋白质中,涉及肿瘤细胞骨架蛋白、糖酵解活动及分子伴侣等方面,揭示宫颈癌TP新辅助化疗影响了多个蛋白质的表达,TP可能是通过调节这些蛋白质功能来起作用的。
3、Western blot验证结果显示HSP70、HSP27、烯醇化酶1、醛缩酶A在化疗前组中的表达高于化疗后组,与蛋白质组学研究结果一致。这些蛋白可能是抗癌药物靶点,下调其表达可能增加宫颈癌对TP的药物敏感性,有望成为临床治疗新靶点,改善TP疗效。
Objective:To establish 2-DE patterns of cervical cancer pre-and post-neoadjvant chemotherapy with paclitaxel and cisplatin in sensitive cases and to find canditate proteins correlating to TP for improving effects of cervical cancer neoadjuvant chemotherapy with TP or finding new anti-cancer drugs.
Methods:1. Six cervical cancer patients were selected which have the same characterization:①locally advanced cauliflower-type before chemotherapy;②neoadjuvant chemotherapy with paclitaxel plus cisplatin:all patients had once chemotherapy, paclitaxel 175mg/m2, cisplatin 60mg/m2;③Evaluation of efficacy of chemotherapy in 10 days were partial remission(PR), tumor size decreased 50% or more;④Radical hysterectomy and pelvic lymph node dissection were carried out about 15 days after chemotherapy;⑤Pathological diagnosis after operation were moderately differentiated squamous cell carcinoma. Samples were immediately conserved in -80℃after collection pre- and post- neoadjvant chemotherapy. Protein samples extracted from tissues were isolated by 2-D electrophoresis. The images obtained by Coomassie brilliant blue staining and scanning images were analyzed with PD-quest 7.0 software to identify differentially expressed protein spots between the two groups.
2.According to the proteins classification and the degree of difficulties for buying antibodies, four differentially expressed proteins were selected for verification. By Western blot methods the expressions of four different proteins were observed pre-and post-chemotherapy in the six cases of cervical cancer tissue to detect whether the results are consistent with the proteomics. The protein were extracted from the cervical cancer tissues as proteomics study did. The same amount of sample were put to PAGE gel after measuring protein concentration. Then SDS-PAGE electrophoresis, PVDF transfer membrane, nonfat dry milk closed, and antibody incubation, HRP-ECL luminescence color, Fuser film, scanning and analysis were made.GAPDH as an internal reference.
Results:1.The high-resolution, good reproducible 2-DE images were established.598±64 and 673±79 protein spots were found respectively pre- and post- chemotherapy, the average match rate between groups were 85.4%. Differentially expressed proteins between the two groups points were 102. Thirteen points significantly differentially expressed were selected for MS analysis. All candidate proteins were identified successfully. Compared with pre- chemotherapy, the expression of 11 proteins were downregulated and 2 proteins were upregulated in the post- chemotherapy group. Downregulated proteins include:actinin al,lamin Bl,eukaryotic translation elongation factor 1γ, enolase 1, annexin 1, epithelial cell marker protein 1, aldolase A, heat shock protein 27, pyruvate kinase, keratinⅡ-type and heat shock protein 70; Upregulated proteins include:apolipoprotein A1 and annexin V. All these proteins can be categorized into three groups:①Cytoskeletal proteins:actininα1, lamin B1, keratin typeⅡ, annexinⅠ, annexinⅤ;②Glycolytic enzymes:enolase 1, aldolase A, pyruvate kinase;③Others:eukaryotic translation elongation factor 1γ, epithelial cell marker protein 1, HSP27, HSP70 and apolipoprotein Al.
2. The expression of HSP27, HSP70, enolase 1, aldolase A were conformed by Western blot method and results showed that the four proteins in post- chemotherapy group have lower expression than pre- chemotherapy group. The results were consistent with the proteomics study.
Conclusion:1. The establishment of two-dimensional electrophoresis images for pre- and post- neoadjuvant chemotherapy with paclitaxel and cisplatin of cervical cancer show that the TP can lead to changes in the protein expression profile of cervical cancer.
2. TP in chemotherapy of cervical cancer may play its roles by regulating cytoskeletal proteins, HSP, glycolytic enzymes and other proteins.
3. Western blot results showed that the expression of HSP70, HSP27, enolase 1, aldolase A in post-chemotherapy group were lower than the pre-chemotherapy group and it was consistent with proteomics results. More study of these proteins may be expected to find new anticancer drug targets or to improve the efficacy of TP by down-regulating them.
采用双向凝胶电泳(two-dimensional gelelectrophoresis,2-DE)结合质谱分析技术比较对紫杉醇(paclitaxel, T)联合顺铂(cisplatin, P)新辅助化疗作用敏感的宫颈癌组织化疗前后蛋白质组的差异,建立TP化疗前后的宫颈癌2—DE图谱,识别和鉴定差异表达的蛋白质,初步确定宫颈癌中与TP作用相关的蛋白质,研究其作用机制,为改善宫颈癌患者TP新辅助化疗疗效、研制新的肿瘤靶向药物提供实验及理论依据。
方法：
1、选取6例宫颈癌住院病人,纳入标准为：①化疗前为局部晚期菜花型；②采用紫杉醇加顺铂新辅助化疗：均化疗1次,紫杉醇175mg／m2,顺铂60mg／m2；③化疗10天后评价疗效为显效,肿瘤体积缩小50%以上；④约化疗后15天行广泛性子宫切除术及盆腔淋巴结清扫术；⑤术后病理诊断均为中分化鳞癌。宫颈癌组织化疗前后标本收集后立即-80℃保存。提取组织总蛋白,采用2-DE分离蛋白质,经考马斯亮蓝染色、图像扫描,得到化疗前后的双向电泳图谱,采用PD-quest7.0软件进行匹配和差异分析,识别两组之间表达差异蛋白点。从胶中切取表达差异明显的蛋白点,用MALDI—TOF—TOF—MS方法进行质谱分析,获取质谱图,数据库搜索鉴定蛋白质。
2、根据获得的差异蛋白的分类及抗体购买的难易程度选择4个差异表达蛋白,应用Westem Blot方法进行验证,检测其在以上6例宫颈癌组织标本中化疗前后的表达变化,观察它们在化疗前后的表达是否与蛋白质组学方法检测结果一致。以蛋白质组学所用提取的组织蛋白为研究对象,制备PAGE胶,测蛋白浓度后等量上样,SDS-PAGE电泳,PVDF转膜,脱脂奶粉封闭,抗体孵育,HRP-ECL发光法显色,胶片定影、扫描、分析。以GAPDH为内参。
结果：
1、建立了分辨率高、重复性好的宫颈癌化疗前后的双向凝胶电泳图谱。化疗前蛋白点598士64个,化疗后蛋白点673士79个,组间平均匹配率为85.4%。
2、两组间差异表达蛋白质点数为102个,从中选择表达差异明显的13个点进行质谱分析,全部鉴定成功,其中化疗后与化疗前相比,11个蛋白下调,2个蛋白上调。化疗后表达下调的蛋白：辅肌动蛋白α1(actinin alpha 1)、核纤层蛋白B1 (lamin B1)、真核翻译延长因子1γ(Eukaryotic translation elongation factor 1 gamma)、烯醇化酶1(enolase 1)、膜联蛋白1 (annexinⅠ)、上皮细胞标记蛋白1(epithelial cell marker protein 1)、醛缩酶A(aldolase A)、热休克蛋白27(heat shock protein 27)、丙酮酸激酶(pyruvate kinase)、角蛋白Ⅱ型(cytokeratin typeⅡ)、热休克蛋白70(heat shock protein 70);化疗后表达上调的蛋白：载脂蛋白A1(apolipoprotein A-1)、膜联蛋白V (annexin V)。差异蛋白可归纳为3类：①细胞骨架蛋白：辅肌动蛋白α1,核纤层蛋白B1,角蛋白Ⅱ型,膜联蛋白工,膜联蛋白V；②糖酵解酶类：烯醇化酶1,醛缩酶A,丙酮酸激酶；③其它：真核翻译延长因子1γ,上皮细胞标记蛋白1,热休克蛋白27,热休克蛋白70,载脂蛋白Al。
3、选择HSP70、HSP27、烯醇化酶1、醛缩酶A用Western blot技术进行验证,结果显示这四种蛋白在化疗前组中的表达高于化疗后组。该结果与蛋白质组学研究结果一致。
结论：
1、本研究通过提取宫颈癌化疗前后的总蛋白质,进行双向凝胶电泳,建立了宫颈癌紫杉醇联合顺铂新辅助化疗前后的凝胶电泳图谱,并应用PD—quest7.0软件对获取的凝胶图像进行分析,发现差异表达蛋白质。表明TP能够导致人宫颈癌蛋白质表达谱的改变。
2、本研究应用MALDI—TOF—TOF—MS方法分析部分差异表达明显的蛋白质点,获取肽质量指纹图谱,搜索数据库鉴定出了13个差异蛋白质。这些差异表达的蛋白质中,涉及肿瘤细胞骨架蛋白、糖酵解活动及分子伴侣等方面,揭示宫颈癌TP新辅助化疗影响了多个蛋白质的表达,TP可能是通过调节这些蛋白质功能来起作用的。
3、Western blot验证结果显示HSP70、HSP27、烯醇化酶1、醛缩酶A在化疗前组中的表达高于化疗后组,与蛋白质组学研究结果一致。这些蛋白可能是抗癌药物靶点,下调其表达可能增加宫颈癌对TP的药物敏感性,有望成为临床治疗新靶点,改善TP疗效。
Objective:To establish 2-DE patterns of cervical cancer pre-and post-neoadjvant chemotherapy with paclitaxel and cisplatin in sensitive cases and to find canditate proteins correlating to TP for improving effects of cervical cancer neoadjuvant chemotherapy with TP or finding new anti-cancer drugs.
Methods:1. Six cervical cancer patients were selected which have the same characterization:①locally advanced cauliflower-type before chemotherapy;②neoadjuvant chemotherapy with paclitaxel plus cisplatin:all patients had once chemotherapy, paclitaxel 175mg/m2, cisplatin 60mg/m2;③Evaluation of efficacy of chemotherapy in 10 days were partial remission(PR), tumor size decreased 50% or more;④Radical hysterectomy and pelvic lymph node dissection were carried out about 15 days after chemotherapy;⑤Pathological diagnosis after operation were moderately differentiated squamous cell carcinoma. Samples were immediately conserved in -80℃after collection pre- and post- neoadjvant chemotherapy. Protein samples extracted from tissues were isolated by 2-D electrophoresis. The images obtained by Coomassie brilliant blue staining and scanning images were analyzed with PD-quest 7.0 software to identify differentially expressed protein spots between the two groups.
2.According to the proteins classification and the degree of difficulties for buying antibodies, four differentially expressed proteins were selected for verification. By Western blot methods the expressions of four different proteins were observed pre-and post-chemotherapy in the six cases of cervical cancer tissue to detect whether the results are consistent with the proteomics. The protein were extracted from the cervical cancer tissues as proteomics study did. The same amount of sample were put to PAGE gel after measuring protein concentration. Then SDS-PAGE electrophoresis, PVDF transfer membrane, nonfat dry milk closed, and antibody incubation, HRP-ECL luminescence color, Fuser film, scanning and analysis were made.GAPDH as an internal reference.
Results:1.The high-resolution, good reproducible 2-DE images were established.598±64 and 673±79 protein spots were found respectively pre- and post- chemotherapy, the average match rate between groups were 85.4%. Differentially expressed proteins between the two groups points were 102. Thirteen points significantly differentially expressed were selected for MS analysis. All candidate proteins were identified successfully. Compared with pre- chemotherapy, the expression of 11 proteins were downregulated and 2 proteins were upregulated in the post- chemotherapy group. Downregulated proteins include:actinin al,lamin Bl,eukaryotic translation elongation factor 1γ, enolase 1, annexin 1, epithelial cell marker protein 1, aldolase A, heat shock protein 27, pyruvate kinase, keratinⅡ-type and heat shock protein 70; Upregulated proteins include:apolipoprotein A1 and annexin V. All these proteins can be categorized into three groups:①Cytoskeletal proteins:actininα1, lamin B1, keratin typeⅡ, annexinⅠ, annexinⅤ;②Glycolytic enzymes:enolase 1, aldolase A, pyruvate kinase;③Others:eukaryotic translation elongation factor 1γ, epithelial cell marker protein 1, HSP27, HSP70 and apolipoprotein Al.
2. The expression of HSP27, HSP70, enolase 1, aldolase A were conformed by Western blot method and results showed that the four proteins in post- chemotherapy group have lower expression than pre- chemotherapy group. The results were consistent with the proteomics study.
Conclusion:1. The establishment of two-dimensional electrophoresis images for pre- and post- neoadjuvant chemotherapy with paclitaxel and cisplatin of cervical cancer show that the TP can lead to changes in the protein expression profile of cervical cancer.
2. TP in chemotherapy of cervical cancer may play its roles by regulating cytoskeletal proteins, HSP, glycolytic enzymes and other proteins.
3. Western blot results showed that the expression of HSP70, HSP27, enolase 1, aldolase A in post-chemotherapy group were lower than the pre-chemotherapy group and it was consistent with proteomics results. More study of these proteins may be expected to find new anticancer drug targets or to improve the efficacy of TP by down-regulating them.
引文
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