胃癌血管特异性短肽GEBP11受体信息的初步筛选
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摘要
【背景】
肿瘤的血管生成与肿瘤的生长、侵袭、转移和预后有着密切的联系。对肿瘤血管生成的研究为肿瘤的诊断和治疗提供了新的策略和良好前景。然而,由于缺乏肿瘤血管靶向性分子,通过肿瘤血管诊断治疗肿瘤尚未取得令人满意的结果。研究证实,在肿瘤微环境的作用下肿瘤血管内皮细胞的遗传组成受到影响,表达异质性分子[1, 2]。异质性分子的存在将为肿瘤的靶向诊断及治疗提供特异性靶标。肿瘤血管的异质性取决于肿瘤的异质性,由肿瘤的类型及微环境决定[3, 4]。胃癌细胞与血管内皮细胞的体外共培养可以模拟肿瘤微环境,诱导胃癌血管异质性分子的表达。前期工作中,我们通过建立胃癌细胞SGC7901和人脐静脉内皮细胞的体外共培养模型,利用噬菌体随机环七肽库体外筛选获得能特异结合于共培养内皮细胞的短肽GEBP11。通过初步鉴定证实,GEBP11具有良好的胃癌血管靶向性[5]和血管生成抑制活性。筛选GEBP11的结合受体对深入研究GEBP11实现靶向性和血管抑制活性的机制具有重要意义。因此,本课题围绕筛选GEBP11结合受体的相关信息展开研究,旨在为获得GEBP11的结合受体奠定坚实的基础。
【目的】
1.获得GEBP11结合受体在多种胃癌细胞系、多种正常组织和多种器官肿瘤组织血管的表达分布信息;2.研究GEBP11干预后Co-HUVECs分子群的表达变化,鉴定GEBP11影响的信号通路,为受体筛选提供参考信息;3.筛选GEBP11的结合受体,为阐明GEBP11的机制和功能奠定基础。
【方法】
1.通过免疫组织化学染色的方法检测GEBP11在多种正常组织、多器官肿瘤组织血管上的结合特性,通过免疫细胞化学染色的方法检测GEBP11在不同胃癌细胞系上的结合特性;
2.通过RT-PCR和Western blot对基因表达谱分析的结果进行验证,并对基因表达谱分析的结果进行深入的生物信息学分析,鉴定GEBP11影响的信号通路;
3.通过免疫沉淀和质谱分析的方法初步筛选GEBP11的受体。
【结果】
1. GEBP11受体的分布信息
1) GEBP11结合受体在多种细胞系上的表达分布
免疫细胞化学染色结果显示,GEBP11在Co-HUVECs和MKN45染色呈阳性,在GES染色弱阳性,在MKN28、AGS、SGC7901染色呈阴性;URP、PBS对照在各细胞系染色均呈阴性。
2) GEBP11的结合受体在多种器官肿瘤中的表达分布
GEBP11在多器官肿瘤组织的免疫化学染色显示,GEBP11的结合受体在胃癌、胰腺癌组织血管呈强阳性表达;在食管癌、结肠癌、肝细胞肝癌、子宫癌组织血管呈弱阳性表达;在直肠癌、肺癌、膀胱癌组织血管呈阴性表达。上述9种肿瘤对应的癌旁组织中,GEBP11的结合受体在子宫癌和膀胱癌的癌旁组织血管呈阳性表达;其余均为阴性表达。GEBP11的结合受体在肝细胞肝癌、膀胱尿路上皮癌组织呈阳性表达,在慢性结肠粘膜炎和慢性直肠粘膜炎的腺体、肾组织、膀胱粘膜等癌旁组织中呈阳性表达,在其余肿瘤和癌旁组织实质呈阴性表达。
3) GEBP11的结合受体在正常组织中的表达分布
GEBP11在食管、肝、胰腺、垂体、甲状腺、甲状旁腺、心肌、肺、肾、胸腺、骨髓、脾脏、乳腺、卵巢、扁桃体、睾丸、前列腺、涎腺、胃、小肠、结肠等各种正常组织血管染色均为阴性,在食管粘膜细胞、胃腺体、结肠腺体、肾小管、睾丸实质染色阳性。
2. GEBP11干预后Co-HUVECs表达变化的分子群和影响的信号通路
经验证,RT-PCR与Western blot的结果与基因表达谱一致;差异基因筛选、GoTerm和Pathway分析为GEBP11结合受体的筛选提供了信息。
3. GEBP11受体的筛选
通过免疫沉淀-质谱分析的方法筛选获得了12个GEBP11结合受体的候选分子;通过与基因表达谱结果结合分析发现候选蛋白ZCCHC11经GEBP11处理后表达上调约3倍。
【结论】
1. GEBP11的受体表达于一些低分化胃癌细胞系和某些肿瘤组织血管,而在正常组织血管无表达。
2. RT-PCR和Western blot结果与基因表达谱一致,差异基因筛选、GoTerm和Pathway分析为GEBP11结合受体的筛选提供了信息。
3.筛选获得了12个GEBP11结合受体的候选蛋白,其中候选蛋白ZCCHC11经GEBP11处理后表达上调约3倍。
【Background】
Tumor angiogenesis is related closely to growth, invasion, metastatic and prognosis of tumor. The researches of tumor angiogenesis provide new strategy and perspective for the diagnosis and treatment of tumor. However, the status of diagnosis and treatment of tumor by angiogenesis is not satisfactory due to the deficiency of target molecules. Researches indicated that the genetic component of vascular endothelial cells changed in the tumor microenvironment and the endothelial cells expressed heterogeneity molecules, which would probably provide target molecules for diagnosis and therapeutics of tumor. The heterogeneity of tumor vascular depends on that of tumor and decided by the type and microenvironment of tumor. The co-culture of gastric cancer cells and vascular endothelial cells in vitro could simulate tumor microenvironment and induce the expression of the heterogeneity molecules. In previous studies, we obtained a peptide GEBP11 specifically binding to the co-cultured human umbilical vein endothelial cells (Co-HUVECs) through establishing an in vitro co-culture model of gastric cancer cell line SGC7901 and HUVECs and screening in phage display peptides library. Initial studies suggested that GEBP11 peptide has the ability in targeting to gastric cancer vascular and the biological activity in inhibiting angiogenesis. It is important to screen the receptor of GEBP11 for the mechanistic study of the targeting and angiogenesis inhibiting activities of GEBP11. Therefore, the aim of this study is screening the information about the receptor of GEBP11 and setting a foundation for obtaining the receptor.
【Objectives】
1. To obtain the localizing information of the receptor of GEBP11 in gastric cancer cell lines, normal tissues and multiple organ cancers; 2. To detect the changed genes of Co-HUVECs and pathways influenced after treated by GEBP11, which could provide information for screening the receptor; 3. To screen the receptor of GEBP11 and settle a foundation for elucidating the mechanism and function of GEBP11.
【Methods】
1. Immunohistochemistry and immunocytochemistry were performed to detect the expression of the receptor of GEBP11 in normal tissues, multiple organ cancers and gastric cancer cell lines.
2. The result of gene array was verified by RT-PCR and Western blot; bioinformatics was used to analyze the different genes and pathways in Co-HUVECs after interfered by GEBP11.
3. The receptor of GEBP11 was screened by immunoprecipitation and mass spectroscopy.
【Results】
1. The expression of the receptor of GEBP11
1) The expresstion of the receptor of GEBP11 in cell lines.
The result of immunocytochemistry revealed that GEBP11 was stained on the cytoplasm of Co-HUVECs and MKN45, but not MKN28, SGC7901, AGS, GES.
2) The expresstion of the receptor of GEBP11 in multiple organ cancers.
The result of immunohistochemistry showed that GEBP11 was stained on the vascular of gastric cancer, pancreatic cancer and hysterocarcinoma, but not other cancers including cancer of lung, bladder, suprarenal epithelioma, colon, esophagus, rectum, liver.
3) The expresstion of the receptor of GEBP11 in normal tissues.
The result of immunohistochemistry showed that GEBP11 was not stained on the vascular of normal tissues including esophagus, liver, pancreas, hypophysis, thyroid, parathyroid gland, cardiac muscle, lung, kidney, thymus, marrow, spleen, mammary gland, ovary, tonsil, testis, sialaden, stomach, small intestine, colon.
2. The changed genes of Co-HUVECs and pathways after treat by GEBP11
The results of RT-PCR and Western blot were consistent with gene microarray. The screening of differential genes and analysis of GoTerm and pathway provided information for the receptor of GEBP11.
3. The screening of the receptor of GEBP11
We obtained 12 candidant proteins by using the methods of immunoprecipitation and mass spectrography. Compared with the result of gene microarray, we found that the expression of candidant protein ZCCHC11 was upregulated about 3 folds after treated by GEBP11.
【Conclusions】
1. The receptor of GEBP11 expressed in some poorly differentiated gastric cancer cell lines and vascular of some other cancers, but not that of normal tissues.
2. The results of RT-PCR and Western blot were consistent with gene microarray. The screening of differential genes and analysis of GoTerm and pathway provided information for the receptor of GEBP11.
3. We obtained 12 candidant proteins by using the methods of immunoprecipitation and mass spectrography. Candiant protein ZCCHC11 was upregulated about 3folds after treated by GEBP11.
肿瘤的血管生成与肿瘤的生长、侵袭、转移和预后有着密切的联系。对肿瘤血管生成的研究为肿瘤的诊断和治疗提供了新的策略和良好前景。然而,由于缺乏肿瘤血管靶向性分子,通过肿瘤血管诊断治疗肿瘤尚未取得令人满意的结果。研究证实,在肿瘤微环境的作用下肿瘤血管内皮细胞的遗传组成受到影响,表达异质性分子[1, 2]。异质性分子的存在将为肿瘤的靶向诊断及治疗提供特异性靶标。肿瘤血管的异质性取决于肿瘤的异质性,由肿瘤的类型及微环境决定[3, 4]。胃癌细胞与血管内皮细胞的体外共培养可以模拟肿瘤微环境,诱导胃癌血管异质性分子的表达。前期工作中,我们通过建立胃癌细胞SGC7901和人脐静脉内皮细胞的体外共培养模型,利用噬菌体随机环七肽库体外筛选获得能特异结合于共培养内皮细胞的短肽GEBP11。通过初步鉴定证实,GEBP11具有良好的胃癌血管靶向性[5]和血管生成抑制活性。筛选GEBP11的结合受体对深入研究GEBP11实现靶向性和血管抑制活性的机制具有重要意义。因此,本课题围绕筛选GEBP11结合受体的相关信息展开研究,旨在为获得GEBP11的结合受体奠定坚实的基础。
【目的】
1.获得GEBP11结合受体在多种胃癌细胞系、多种正常组织和多种器官肿瘤组织血管的表达分布信息;2.研究GEBP11干预后Co-HUVECs分子群的表达变化,鉴定GEBP11影响的信号通路,为受体筛选提供参考信息;3.筛选GEBP11的结合受体,为阐明GEBP11的机制和功能奠定基础。
【方法】
1.通过免疫组织化学染色的方法检测GEBP11在多种正常组织、多器官肿瘤组织血管上的结合特性,通过免疫细胞化学染色的方法检测GEBP11在不同胃癌细胞系上的结合特性;
2.通过RT-PCR和Western blot对基因表达谱分析的结果进行验证,并对基因表达谱分析的结果进行深入的生物信息学分析,鉴定GEBP11影响的信号通路;
3.通过免疫沉淀和质谱分析的方法初步筛选GEBP11的受体。
【结果】
1. GEBP11受体的分布信息
1) GEBP11结合受体在多种细胞系上的表达分布
免疫细胞化学染色结果显示,GEBP11在Co-HUVECs和MKN45染色呈阳性,在GES染色弱阳性,在MKN28、AGS、SGC7901染色呈阴性;URP、PBS对照在各细胞系染色均呈阴性。
2) GEBP11的结合受体在多种器官肿瘤中的表达分布
GEBP11在多器官肿瘤组织的免疫化学染色显示,GEBP11的结合受体在胃癌、胰腺癌组织血管呈强阳性表达;在食管癌、结肠癌、肝细胞肝癌、子宫癌组织血管呈弱阳性表达;在直肠癌、肺癌、膀胱癌组织血管呈阴性表达。上述9种肿瘤对应的癌旁组织中,GEBP11的结合受体在子宫癌和膀胱癌的癌旁组织血管呈阳性表达;其余均为阴性表达。GEBP11的结合受体在肝细胞肝癌、膀胱尿路上皮癌组织呈阳性表达,在慢性结肠粘膜炎和慢性直肠粘膜炎的腺体、肾组织、膀胱粘膜等癌旁组织中呈阳性表达,在其余肿瘤和癌旁组织实质呈阴性表达。
3) GEBP11的结合受体在正常组织中的表达分布
GEBP11在食管、肝、胰腺、垂体、甲状腺、甲状旁腺、心肌、肺、肾、胸腺、骨髓、脾脏、乳腺、卵巢、扁桃体、睾丸、前列腺、涎腺、胃、小肠、结肠等各种正常组织血管染色均为阴性,在食管粘膜细胞、胃腺体、结肠腺体、肾小管、睾丸实质染色阳性。
2. GEBP11干预后Co-HUVECs表达变化的分子群和影响的信号通路
经验证,RT-PCR与Western blot的结果与基因表达谱一致;差异基因筛选、GoTerm和Pathway分析为GEBP11结合受体的筛选提供了信息。
3. GEBP11受体的筛选
通过免疫沉淀-质谱分析的方法筛选获得了12个GEBP11结合受体的候选分子;通过与基因表达谱结果结合分析发现候选蛋白ZCCHC11经GEBP11处理后表达上调约3倍。
【结论】
1. GEBP11的受体表达于一些低分化胃癌细胞系和某些肿瘤组织血管,而在正常组织血管无表达。
2. RT-PCR和Western blot结果与基因表达谱一致,差异基因筛选、GoTerm和Pathway分析为GEBP11结合受体的筛选提供了信息。
3.筛选获得了12个GEBP11结合受体的候选蛋白,其中候选蛋白ZCCHC11经GEBP11处理后表达上调约3倍。
【Background】
Tumor angiogenesis is related closely to growth, invasion, metastatic and prognosis of tumor. The researches of tumor angiogenesis provide new strategy and perspective for the diagnosis and treatment of tumor. However, the status of diagnosis and treatment of tumor by angiogenesis is not satisfactory due to the deficiency of target molecules. Researches indicated that the genetic component of vascular endothelial cells changed in the tumor microenvironment and the endothelial cells expressed heterogeneity molecules, which would probably provide target molecules for diagnosis and therapeutics of tumor. The heterogeneity of tumor vascular depends on that of tumor and decided by the type and microenvironment of tumor. The co-culture of gastric cancer cells and vascular endothelial cells in vitro could simulate tumor microenvironment and induce the expression of the heterogeneity molecules. In previous studies, we obtained a peptide GEBP11 specifically binding to the co-cultured human umbilical vein endothelial cells (Co-HUVECs) through establishing an in vitro co-culture model of gastric cancer cell line SGC7901 and HUVECs and screening in phage display peptides library. Initial studies suggested that GEBP11 peptide has the ability in targeting to gastric cancer vascular and the biological activity in inhibiting angiogenesis. It is important to screen the receptor of GEBP11 for the mechanistic study of the targeting and angiogenesis inhibiting activities of GEBP11. Therefore, the aim of this study is screening the information about the receptor of GEBP11 and setting a foundation for obtaining the receptor.
【Objectives】
1. To obtain the localizing information of the receptor of GEBP11 in gastric cancer cell lines, normal tissues and multiple organ cancers; 2. To detect the changed genes of Co-HUVECs and pathways influenced after treated by GEBP11, which could provide information for screening the receptor; 3. To screen the receptor of GEBP11 and settle a foundation for elucidating the mechanism and function of GEBP11.
【Methods】
1. Immunohistochemistry and immunocytochemistry were performed to detect the expression of the receptor of GEBP11 in normal tissues, multiple organ cancers and gastric cancer cell lines.
2. The result of gene array was verified by RT-PCR and Western blot; bioinformatics was used to analyze the different genes and pathways in Co-HUVECs after interfered by GEBP11.
3. The receptor of GEBP11 was screened by immunoprecipitation and mass spectroscopy.
【Results】
1. The expression of the receptor of GEBP11
1) The expresstion of the receptor of GEBP11 in cell lines.
The result of immunocytochemistry revealed that GEBP11 was stained on the cytoplasm of Co-HUVECs and MKN45, but not MKN28, SGC7901, AGS, GES.
2) The expresstion of the receptor of GEBP11 in multiple organ cancers.
The result of immunohistochemistry showed that GEBP11 was stained on the vascular of gastric cancer, pancreatic cancer and hysterocarcinoma, but not other cancers including cancer of lung, bladder, suprarenal epithelioma, colon, esophagus, rectum, liver.
3) The expresstion of the receptor of GEBP11 in normal tissues.
The result of immunohistochemistry showed that GEBP11 was not stained on the vascular of normal tissues including esophagus, liver, pancreas, hypophysis, thyroid, parathyroid gland, cardiac muscle, lung, kidney, thymus, marrow, spleen, mammary gland, ovary, tonsil, testis, sialaden, stomach, small intestine, colon.
2. The changed genes of Co-HUVECs and pathways after treat by GEBP11
The results of RT-PCR and Western blot were consistent with gene microarray. The screening of differential genes and analysis of GoTerm and pathway provided information for the receptor of GEBP11.
3. The screening of the receptor of GEBP11
We obtained 12 candidant proteins by using the methods of immunoprecipitation and mass spectrography. Compared with the result of gene microarray, we found that the expression of candidant protein ZCCHC11 was upregulated about 3 folds after treated by GEBP11.
【Conclusions】
1. The receptor of GEBP11 expressed in some poorly differentiated gastric cancer cell lines and vascular of some other cancers, but not that of normal tissues.
2. The results of RT-PCR and Western blot were consistent with gene microarray. The screening of differential genes and analysis of GoTerm and pathway provided information for the receptor of GEBP11.
3. We obtained 12 candidant proteins by using the methods of immunoprecipitation and mass spectrography. Candiant protein ZCCHC11 was upregulated about 3folds after treated by GEBP11.
引文
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