新型特异性抗Met单克隆抗体用于大肠癌中Met表达检测重组人高尔基体蛋白GP73原核表达、纯化及鉴定
摘要
原癌基因MET编码的蛋白产物Met为肝细胞生长因子(HGF/SF)的受体,具有酪氨酸激酶活性,参与细胞信号传导、细胞骨架重排的调控,是参与细胞增殖、分化和运动的重要因素。多项研究已证实在肿瘤组织中Met的过表达与肿瘤的浸润、转移和不良预后密切相关。但在大肠癌组织中Met蛋白的表达水平与大肠癌的临床分期,病理分级,生存期等关系的研究结果尚存争议,可能与目前尚无专门用于检测福尔马林固定标本中Met表达水平的特异性单克隆抗体有关。在我们前期研究中已成功建立了一株鼠源性抗Met单克隆抗体Met4,可以特异性结合福尔马林处理过的Met受体胞外区。在对多种人类肿瘤细胞的免疫组化,免疫荧光染色,Western blot实验结果的分析均显示,Met4特异性,敏感性以及可重复性优于市场上其他抗Met抗体。
目的
1.观察研究Met在大肠癌组织中的表达水平及其与临床病理特征的关系,探讨其表达改变与肿瘤发生、侵袭、转移的关系,为大肠癌的预后判断及临床靶向治疗提供依据。
2.比较分析新型特异性抗Met单克隆抗体Met4和国际通用兔抗Met多克隆抗体C28在检测大肠癌蜡块组织中Met表达水平的差别。
方法
采用免疫组织化学染色方法检测78例不同临床分期及病理分级大肠癌组织及6例大肠腺瘤组织中Met表达水平,分析Met的表达与大肠癌临床病理特征的关系。进一步采用荧光免疫组化方法比较分析Met4与C28的检测效能。
结果
1. 78例大肠癌组织中Met细胞浆染色结果:强阳性46例,弱阳性22例,阳性表达率分别为59%,28.2%;阴性10例(12.8%),即肿瘤细胞浆及细胞膜上均未出现棕黄色染色。6例大肠腺瘤组织,强阳性2例,弱阳性2例,阴性2例。切缘正常大肠组织中Met表达阴性或弱阳性。Met细胞浆阳性表达率在不同病理分级,不同临床分期的差异无明显统计学意义。
2. 78例大肠癌组织中细胞膜Met染色结果:将细胞膜染色评分3分的病例定义为Met细胞膜高表达组,细胞膜染色评分≤2分病例定义为Met细胞膜表达低表达组。两位阅片人双盲评分得出:淋巴转移组Met膜阳性表达率高于淋巴未转移组,且肿瘤细胞Met膜阳性更多的出现在临床分期Ⅰ期和Ⅲ期的病例中,临床分期Ⅱ期和Ⅳ期几乎无Met膜阳性,只表现Met浆阳性。
3. Met4和C28在免疫组化染色及荧光免疫组化染色中,对Met的亚细胞定位存在差异。使用Met4检测时Met定位在肿瘤细胞浆和/或细胞膜,细胞核无染色。C28检测时Met定位在肿瘤细胞核。
结论
细胞膜高表达Met的大肠癌病例淋巴转移率明显增高,预示了肿瘤临床分级及不良预后。因此Met可作为反映大肠癌生物学行为的参考指标,有助于大肠癌的预后判断,并提供肿瘤治疗的干预靶点。Met4可以特异性结合福尔马林处理过的Met受体胞外区,用于筛选适宜于靶向阻断Met信号通路治疗的肿瘤患者,具有良好的临床应用前景。
The proto-oncogene MET, which encodes the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met, is frequently expressed in the surface of epithelial cells. The tyrosine kinase receptor Met together with its ligand HGF/SF regulates cellular proliferation, migration and differentiation during organ development and tissue homeostasis. Overexpression of Met has been shown to be associated with poor prognosis in a number of solid tumors. In colorectal cancer(CRC), however, the relationship between Met protein level and clinical character has been controversial, which was mostly determined by the lackage of the reliable Met antibody for evaluating Met expression. In our previous study, a novel mouse monoclonal antibody Met4 was developed and validated to specifically reacts with an epitope in the extracellular domain of Met in FFPE tissues. In immunohistochemistry (IHC), immunofluorescence and Western blot analysis, Met4 may react strongly with Met as expressed on the cultured human cancer cells including human CRC cells. Met4 can be worked as a useful reagent for reproducible, specific and sensitive quantification of Met protein expression levels in all fixed mammalian cells.
Objective
To investigate the expression level of Met in human colorectal cancer and the relationship between Met expression levels and clinicopathological factors, and explore the effect of Met in the process of invasion and metastasis in CRC.
To compare the specificity of Met4 and commercial rabbit anti human Met polyclonal antibody C28(sc-161, Santa Cruz, USA) for detecting the formalin-treated Met protein in the paraffin-embedded CRC specimens.
Methods
The expression levels of Met in formalin-fixed and paraffin-embedded (FFPB) specimens from 78 primary CRC and 6 adenoma were estimated by IHC using Met4 as well as the commercial antibody C28. The relationship between Met expression levels and clinicopathological factors was evaluated. Simultaneously we performed fluorescent-based IHC to compare the reactivity efficacy of Met4 in the colorectal cancer with that of the polyclonal antibody C28.
Results
1. Met cytoplasm staining results of 78 primary CRC: positive, 46 cases; moderate, 22 cases (the rates of positive expression were 59% and 28.2%, respectively); negative, 10 cases (12.8%). Met cytoplasm staining results of 6 adenoma: positive, 2 cases; moderate, 2 cases; negtive, 2 cases. Normal colon epithelium cells showed negative or weak cytoplasm staining. No significant statistical differences were observed in the cytoplasm staining intensity across clinical subtypes of colorectal cancer.
2. Met membrane staining results of 78 primary CRC: When membrane immunostaining score 3 was considered as positive and≤2 as negative, Met membrane expression may be associated with the lymphatic early-stage invasion and metastasis of CRC. stageⅢand stageⅠwere tend to present higher membrane Met staining than cytoplasm Met staining, while the cytoplasm Met staining were mostly appeared in stageⅡand stageⅣpatients.
3. In chemochromagen and fluorescent-based IHC analysis by Met4, Met staining exhibited a combined membranous and cytoplasmic pattern, and nuclear staining was not observed. While in the analysis by polyclonal antibody C28, Met staining revealed positive brown signals in nuclei of tumor cells, and fluorescent-based IHC results showed there was non-specificity response with C28.
Conclusion
The data from this study demonstrated the specificity and reproducibility of Met4 for assessing Met in CRC primary tumor. The membrane distribution of Met in CRC primary tumor may predict tumor regional metastatic potential. The clinical application of Met4 antibody may be explored to select the potential responders who may be benefit from Met antagonistic drugs.
目的
1.观察研究Met在大肠癌组织中的表达水平及其与临床病理特征的关系,探讨其表达改变与肿瘤发生、侵袭、转移的关系,为大肠癌的预后判断及临床靶向治疗提供依据。
2.比较分析新型特异性抗Met单克隆抗体Met4和国际通用兔抗Met多克隆抗体C28在检测大肠癌蜡块组织中Met表达水平的差别。
方法
采用免疫组织化学染色方法检测78例不同临床分期及病理分级大肠癌组织及6例大肠腺瘤组织中Met表达水平,分析Met的表达与大肠癌临床病理特征的关系。进一步采用荧光免疫组化方法比较分析Met4与C28的检测效能。
结果
1. 78例大肠癌组织中Met细胞浆染色结果:强阳性46例,弱阳性22例,阳性表达率分别为59%,28.2%;阴性10例(12.8%),即肿瘤细胞浆及细胞膜上均未出现棕黄色染色。6例大肠腺瘤组织,强阳性2例,弱阳性2例,阴性2例。切缘正常大肠组织中Met表达阴性或弱阳性。Met细胞浆阳性表达率在不同病理分级,不同临床分期的差异无明显统计学意义。
2. 78例大肠癌组织中细胞膜Met染色结果:将细胞膜染色评分3分的病例定义为Met细胞膜高表达组,细胞膜染色评分≤2分病例定义为Met细胞膜表达低表达组。两位阅片人双盲评分得出:淋巴转移组Met膜阳性表达率高于淋巴未转移组,且肿瘤细胞Met膜阳性更多的出现在临床分期Ⅰ期和Ⅲ期的病例中,临床分期Ⅱ期和Ⅳ期几乎无Met膜阳性,只表现Met浆阳性。
3. Met4和C28在免疫组化染色及荧光免疫组化染色中,对Met的亚细胞定位存在差异。使用Met4检测时Met定位在肿瘤细胞浆和/或细胞膜,细胞核无染色。C28检测时Met定位在肿瘤细胞核。
结论
细胞膜高表达Met的大肠癌病例淋巴转移率明显增高,预示了肿瘤临床分级及不良预后。因此Met可作为反映大肠癌生物学行为的参考指标,有助于大肠癌的预后判断,并提供肿瘤治疗的干预靶点。Met4可以特异性结合福尔马林处理过的Met受体胞外区,用于筛选适宜于靶向阻断Met信号通路治疗的肿瘤患者,具有良好的临床应用前景。
The proto-oncogene MET, which encodes the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met, is frequently expressed in the surface of epithelial cells. The tyrosine kinase receptor Met together with its ligand HGF/SF regulates cellular proliferation, migration and differentiation during organ development and tissue homeostasis. Overexpression of Met has been shown to be associated with poor prognosis in a number of solid tumors. In colorectal cancer(CRC), however, the relationship between Met protein level and clinical character has been controversial, which was mostly determined by the lackage of the reliable Met antibody for evaluating Met expression. In our previous study, a novel mouse monoclonal antibody Met4 was developed and validated to specifically reacts with an epitope in the extracellular domain of Met in FFPE tissues. In immunohistochemistry (IHC), immunofluorescence and Western blot analysis, Met4 may react strongly with Met as expressed on the cultured human cancer cells including human CRC cells. Met4 can be worked as a useful reagent for reproducible, specific and sensitive quantification of Met protein expression levels in all fixed mammalian cells.
Objective
To investigate the expression level of Met in human colorectal cancer and the relationship between Met expression levels and clinicopathological factors, and explore the effect of Met in the process of invasion and metastasis in CRC.
To compare the specificity of Met4 and commercial rabbit anti human Met polyclonal antibody C28(sc-161, Santa Cruz, USA) for detecting the formalin-treated Met protein in the paraffin-embedded CRC specimens.
Methods
The expression levels of Met in formalin-fixed and paraffin-embedded (FFPB) specimens from 78 primary CRC and 6 adenoma were estimated by IHC using Met4 as well as the commercial antibody C28. The relationship between Met expression levels and clinicopathological factors was evaluated. Simultaneously we performed fluorescent-based IHC to compare the reactivity efficacy of Met4 in the colorectal cancer with that of the polyclonal antibody C28.
Results
1. Met cytoplasm staining results of 78 primary CRC: positive, 46 cases; moderate, 22 cases (the rates of positive expression were 59% and 28.2%, respectively); negative, 10 cases (12.8%). Met cytoplasm staining results of 6 adenoma: positive, 2 cases; moderate, 2 cases; negtive, 2 cases. Normal colon epithelium cells showed negative or weak cytoplasm staining. No significant statistical differences were observed in the cytoplasm staining intensity across clinical subtypes of colorectal cancer.
2. Met membrane staining results of 78 primary CRC: When membrane immunostaining score 3 was considered as positive and≤2 as negative, Met membrane expression may be associated with the lymphatic early-stage invasion and metastasis of CRC. stageⅢand stageⅠwere tend to present higher membrane Met staining than cytoplasm Met staining, while the cytoplasm Met staining were mostly appeared in stageⅡand stageⅣpatients.
3. In chemochromagen and fluorescent-based IHC analysis by Met4, Met staining exhibited a combined membranous and cytoplasmic pattern, and nuclear staining was not observed. While in the analysis by polyclonal antibody C28, Met staining revealed positive brown signals in nuclei of tumor cells, and fluorescent-based IHC results showed there was non-specificity response with C28.
Conclusion
The data from this study demonstrated the specificity and reproducibility of Met4 for assessing Met in CRC primary tumor. The membrane distribution of Met in CRC primary tumor may predict tumor regional metastatic potential. The clinical application of Met4 antibody may be explored to select the potential responders who may be benefit from Met antagonistic drugs.
引文
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