高尔基体膜蛋白73蛋白质的表达及其在肝癌发生发展中的作用
摘要
我国是肝癌大国,目前最常用的肝癌筛查指标是血清中甲胎蛋白(AFP)水平,然而其敏感性不高,北京协和医院的肝细胞癌患者的AFP敏感性仅为45.8%(＞200ng/ml)。近年来,美国一组科学家发现高尔基体蛋白73(GP73)有可能成为更好的诊断肝癌尤其是早期肝癌的血清标志物。我们的初步研究提示GP73诊断临床肝癌的敏感性和特异性分别高达77%和93%,可以作为肝癌早期诊断的标志物。但GP73在肝癌的发生和发展中的作用需要阐明。
为了制备临床大规模检测GP73所需的抗体诊断试剂盒,我们首先要表达GP73抗原。本实验第一部分首先将GP73 cDNA分成N端和C端进行扩增,并将这两段目的基因连接到原核表达载体pET29a+中,成功构建了两种原核表达载体pET29a+-GP73 N和pET29a+-GP73 C重组质粒,并将其转化到原核表达大肠杆菌Rosetta(DE3)中获得GP73-N/C重组工程菌。转化菌株经诱导后表达GP73-N端和C端蛋白,目前经亲和层析纯化后正在注入小鼠和鸡体内用于制备单克隆和多克隆抗体,从而为临床常规检测GP73奠定了基础。
为了探讨GP73和肝癌发生发展之间的关系,本实验第二部分首先扩增了人GP73基因,连接到逆转录病毒载体pLXIN-hyg中,成功构建了pLXIN-Hyg-GP73重组质粒,将其转染病毒包装细胞PT67,然后在低表达GP73的肝癌细胞系HepG2中,通过稳定病毒转染技术使之过量表达GP73,通过细胞计数发现GP73有促进细胞增殖的能力,提示GP73可能参与肝癌发生发展。
Hepatocellular carcinoma (HCC) is one of the deadest cancers and half of the HCC patients are in China. Although serumα-fetoprotein (AFP) is the only reliable laboratory test for HCC diagnosis, its sensitivity is just around 45.8% (>200ng/ml) for the HCC patients at Peking Union Hospital. Recently, a group of US scientist reported that GP73 could be a better tumor marker for HCC. Our study suggests that the sensitivity and specificity of serum GP73 for HCC patients are 77% and 93%, respectively. However, the causative relationship between GP73 and liver cancer remains to be determined.
To develop a diagnostic kit of GP73 for the screening of HCC patients, recombinant N-terminal and C-terminal GP73 proteins were produced to immunize animals for GP73 antibody. We first PCR amplified GP73 cDNA, then inserted the two cDNA fragments into pET29a+ plasmids to generate pET29a+-GP73N and pET29a+-GP73C vectors for the expression of his-tagged recombinant GP73 proteins. The GP73 proteins that were produced in E.coli Rosetta DE3 were purified for injection of mice and chicken to develop GP73 monoclonal and polyclonal antibodies.
To delineate the role of GP73 in liver tumorigenesis, we took genetic approach to overexpress GP73 in GP73 low-expression liver cancer cell lines. Human GP73 cDNA was PCR amplified, digested, and then inserted into a retroviral expression vector pLXIN-Hyg. The pLXIN-Hyg-GP73 plasmids were transfected into retroviral packaging cell line PT67 cells to produce pLXIN-Hyg-GP73 virus. HepG2 cells were transduced with either pLXIN-Hyg-GP73 virus or pLXIN-Hyg control virus. Since the GP73 over-expressing HepG2 cells grow faster than control cells, we suggest that GP73 may promote hepatic tumorgenesis.
为了制备临床大规模检测GP73所需的抗体诊断试剂盒,我们首先要表达GP73抗原。本实验第一部分首先将GP73 cDNA分成N端和C端进行扩增,并将这两段目的基因连接到原核表达载体pET29a+中,成功构建了两种原核表达载体pET29a+-GP73 N和pET29a+-GP73 C重组质粒,并将其转化到原核表达大肠杆菌Rosetta(DE3)中获得GP73-N/C重组工程菌。转化菌株经诱导后表达GP73-N端和C端蛋白,目前经亲和层析纯化后正在注入小鼠和鸡体内用于制备单克隆和多克隆抗体,从而为临床常规检测GP73奠定了基础。
为了探讨GP73和肝癌发生发展之间的关系,本实验第二部分首先扩增了人GP73基因,连接到逆转录病毒载体pLXIN-hyg中,成功构建了pLXIN-Hyg-GP73重组质粒,将其转染病毒包装细胞PT67,然后在低表达GP73的肝癌细胞系HepG2中,通过稳定病毒转染技术使之过量表达GP73,通过细胞计数发现GP73有促进细胞增殖的能力,提示GP73可能参与肝癌发生发展。
Hepatocellular carcinoma (HCC) is one of the deadest cancers and half of the HCC patients are in China. Although serumα-fetoprotein (AFP) is the only reliable laboratory test for HCC diagnosis, its sensitivity is just around 45.8% (>200ng/ml) for the HCC patients at Peking Union Hospital. Recently, a group of US scientist reported that GP73 could be a better tumor marker for HCC. Our study suggests that the sensitivity and specificity of serum GP73 for HCC patients are 77% and 93%, respectively. However, the causative relationship between GP73 and liver cancer remains to be determined.
To develop a diagnostic kit of GP73 for the screening of HCC patients, recombinant N-terminal and C-terminal GP73 proteins were produced to immunize animals for GP73 antibody. We first PCR amplified GP73 cDNA, then inserted the two cDNA fragments into pET29a+ plasmids to generate pET29a+-GP73N and pET29a+-GP73C vectors for the expression of his-tagged recombinant GP73 proteins. The GP73 proteins that were produced in E.coli Rosetta DE3 were purified for injection of mice and chicken to develop GP73 monoclonal and polyclonal antibodies.
To delineate the role of GP73 in liver tumorigenesis, we took genetic approach to overexpress GP73 in GP73 low-expression liver cancer cell lines. Human GP73 cDNA was PCR amplified, digested, and then inserted into a retroviral expression vector pLXIN-Hyg. The pLXIN-Hyg-GP73 plasmids were transfected into retroviral packaging cell line PT67 cells to produce pLXIN-Hyg-GP73 virus. HepG2 cells were transduced with either pLXIN-Hyg-GP73 virus or pLXIN-Hyg control virus. Since the GP73 over-expressing HepG2 cells grow faster than control cells, we suggest that GP73 may promote hepatic tumorgenesis.
引文
[1]Kladney RD,Bulla GA,Guo L,et al.GP73,a novel golgi-localized protein upregulated by viral infection[J].Gene,2000,249:53-65.
[2]Kladney RD,Cui X,Bulla GA,et al.Expression of GP73,a resident golgi membrane protein,in viral and nonviral liver disease[J].Hepatology,2002,35:1431-1440.
[3]Iftikhar R,Kladney RD,Havlioglu N,et al.Disease- and cell-specific expression of GP73 in human liver disease[J].Am J Gastroenterol,2004,99:1087-1095.
[4]Maitra A,Thuluvath PJ.GP73 and liver disease:a(Golgi) complex enigma[J].Am J Gastroenterol,2004,99:1096-1098.
[5]Block TM,Comunale MA,Lowman M,et al.Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans[J].Proc Natl Acad Sci U S A,2005,102:779-784.
[6]Marrero JA,Romano PR,Nikolaeva O,et al.GP73,a resident golgi glycoprotein,is a novel serum marker for hepatocellular carcinoma[J].J Hepatol,2005,43:1007-1012.
[7]Drake RR,Schwegler EE,Malik G,et al.Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers[J].Mol Cell Proteomics,2006,5:1957-1967.
[8]Cassandra Willyard,Researchers look for 'sweet' method to diagnose cancer[J].Nature Medicine,2007,13:1267.
[9]徐海峰,杨华瑜,毛一雷等.改变肝癌早期诊断和治疗现状的新肝癌血清标志物,基础医学与临床,2008,28:104-108;
[10]毛一雷,杨华瑜,徐海峰等.新的肝癌血清标记物GP73在肝癌诊断 中的初步研究,中华医学杂志,2008,28:104-108;
[1]Taketa K,Okada S,Win N,et al.Evaluation of tumor markers for the detection of hepatocellular carcinoma in Yangon General Hospital,Myanmar[J].Acta Med Okayama,2002,56:317-320.
[2]Marrero JA,Su GL,WeiW,et al.Des-gamma carboxyprothrombincan differentiate hepatocellular carcinoma from non-malignant chronic liver disease in American patients[J].Hepatology,2003,37:1114-1121.
[3]CapurroM,Wanless IR,ShermanM,et al.Glyp ican23:a novel serum and histochemical marker for hepatocellular carcinoma[J].Gastroenterology,2003,125:89-97.
[4]Yamagamim H,MoriyamaM,Matsumura H,et al.Serum concentration of human hepatocyte growth factor is a useful indicator for p redicting the occurrence of hepatocellular carcinomas in C2viral chronic liver diseases[J].Cancer,2002,95:824-834.
[5]Kladney RD,Bulla GA,Guo L,et al.GP73,a novel golgi-localized protein upregulated by viral infection[J].Gene,2000,249:53-65.
[6]Kladney RD,Cui X,Bulla GA,et al.Expression of GP73,a resident golgi membrane protein,in viral and nonviral liver disease[J].Hepatology,2002,35:1431-1440.
[7]Iftikhar R,Kladney RD,Havlioglu N,et al.Disease- and cell-specific expression of GP73 in human liver disease[J].Am J Gastroenterol,2004,99:1087-1095.
[8]Maitra A,Thuluvath PJ.GP73 and liver disease:a(Golgi)complex enigma[J].Am J Gastroenterol,2004,99:1096-1098.
[9]Block TM,Comunale MA,Lowman M,et al.Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans[J].Proc Natl Acad Sci U S A,2005,102:779-784.
[10]Marrero JA,Romano PR,Nikolaeva O,et al.GP73,a resident golgi glycoprotein,is a novel serum marker for hepatocellular carcinoma[J].J Hepatol,2005,43:1007-1012.
[11]Drake RR,Schwegler EE,Malik G,et al.Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers[J].Mol Cell Proteomics,2006,5:1957-1967.
[12]徐海峰,杨华瑜,毛一雷等.改变肝癌早期诊断和治疗现状的新肝癌血清标志物,基础医学与临床,2008,28:104-108;
[13]毛一雷,杨华瑜,徐海峰等.新的肝癌血清标记物GP73在肝癌诊断中的初步研究,中华医学杂志,2008,28:104-108;
[14]Cassandra Willyard,Researchers look for 'sweet' method to diagnose cancer[J].Nature Medicine,2007,13:1267.
[2]Kladney RD,Cui X,Bulla GA,et al.Expression of GP73,a resident golgi membrane protein,in viral and nonviral liver disease[J].Hepatology,2002,35:1431-1440.
[3]Iftikhar R,Kladney RD,Havlioglu N,et al.Disease- and cell-specific expression of GP73 in human liver disease[J].Am J Gastroenterol,2004,99:1087-1095.
[4]Maitra A,Thuluvath PJ.GP73 and liver disease:a(Golgi) complex enigma[J].Am J Gastroenterol,2004,99:1096-1098.
[5]Block TM,Comunale MA,Lowman M,et al.Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans[J].Proc Natl Acad Sci U S A,2005,102:779-784.
[6]Marrero JA,Romano PR,Nikolaeva O,et al.GP73,a resident golgi glycoprotein,is a novel serum marker for hepatocellular carcinoma[J].J Hepatol,2005,43:1007-1012.
[7]Drake RR,Schwegler EE,Malik G,et al.Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers[J].Mol Cell Proteomics,2006,5:1957-1967.
[8]Cassandra Willyard,Researchers look for 'sweet' method to diagnose cancer[J].Nature Medicine,2007,13:1267.
[9]徐海峰,杨华瑜,毛一雷等.改变肝癌早期诊断和治疗现状的新肝癌血清标志物,基础医学与临床,2008,28:104-108;
[10]毛一雷,杨华瑜,徐海峰等.新的肝癌血清标记物GP73在肝癌诊断 中的初步研究,中华医学杂志,2008,28:104-108;
[1]Taketa K,Okada S,Win N,et al.Evaluation of tumor markers for the detection of hepatocellular carcinoma in Yangon General Hospital,Myanmar[J].Acta Med Okayama,2002,56:317-320.
[2]Marrero JA,Su GL,WeiW,et al.Des-gamma carboxyprothrombincan differentiate hepatocellular carcinoma from non-malignant chronic liver disease in American patients[J].Hepatology,2003,37:1114-1121.
[3]CapurroM,Wanless IR,ShermanM,et al.Glyp ican23:a novel serum and histochemical marker for hepatocellular carcinoma[J].Gastroenterology,2003,125:89-97.
[4]Yamagamim H,MoriyamaM,Matsumura H,et al.Serum concentration of human hepatocyte growth factor is a useful indicator for p redicting the occurrence of hepatocellular carcinomas in C2viral chronic liver diseases[J].Cancer,2002,95:824-834.
[5]Kladney RD,Bulla GA,Guo L,et al.GP73,a novel golgi-localized protein upregulated by viral infection[J].Gene,2000,249:53-65.
[6]Kladney RD,Cui X,Bulla GA,et al.Expression of GP73,a resident golgi membrane protein,in viral and nonviral liver disease[J].Hepatology,2002,35:1431-1440.
[7]Iftikhar R,Kladney RD,Havlioglu N,et al.Disease- and cell-specific expression of GP73 in human liver disease[J].Am J Gastroenterol,2004,99:1087-1095.
[8]Maitra A,Thuluvath PJ.GP73 and liver disease:a(Golgi)complex enigma[J].Am J Gastroenterol,2004,99:1096-1098.
[9]Block TM,Comunale MA,Lowman M,et al.Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans[J].Proc Natl Acad Sci U S A,2005,102:779-784.
[10]Marrero JA,Romano PR,Nikolaeva O,et al.GP73,a resident golgi glycoprotein,is a novel serum marker for hepatocellular carcinoma[J].J Hepatol,2005,43:1007-1012.
[11]Drake RR,Schwegler EE,Malik G,et al.Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers[J].Mol Cell Proteomics,2006,5:1957-1967.
[12]徐海峰,杨华瑜,毛一雷等.改变肝癌早期诊断和治疗现状的新肝癌血清标志物,基础医学与临床,2008,28:104-108;
[13]毛一雷,杨华瑜,徐海峰等.新的肝癌血清标记物GP73在肝癌诊断中的初步研究,中华医学杂志,2008,28:104-108;
[14]Cassandra Willyard,Researchers look for 'sweet' method to diagnose cancer[J].Nature Medicine,2007,13:1267.