目的:探讨DKK-1、DKK-2和人磷脂酰肌醇蛋白聚糖3(glypican3,GPC3)蛋白在肝细胞癌组织中的表达及临床意义。方法:采用组织芯片联合免疫组织化学法检测DKK-1、DKK-2和GPC3蛋白在10例正常肝、12例肝硬化、57例肝细胞癌及癌旁肝组织中的表...目的:探讨DKK-1、DKK-2和人磷脂酰肌醇蛋白聚糖3(glypican3,GPC3)蛋白在肝细胞癌组织中的表达及临床意义。方法:采用组织芯片联合免疫组织化学法检测DKK-1、DKK-2和GPC3蛋白在10例正常肝、12例肝硬化、57例肝细胞癌及癌旁肝组织中的表达差异,并分析其临床意义。结果:免疫组织化学检测结果发现,DKK-1和DKK-2在肝细胞癌组织中阳性染色率分别为59.65%(34/57)和57.89%(33/57);GPC3只在肝细胞癌组织中表达,其阳性染色率为47.37%(27/57),而在正常肝、肝硬化及癌旁肝组织中均呈阴性表达。DKK-1与DKK-2阳性表达之间存在密切相关性(χ2=0.570,P=0.000),DKK-2与GPC3阳性染色之间也存在相关性(χ2=0.272,P=0.041)。统计分析DKK-1、DKK-2及GPC3在肝细胞癌组织中阳性染色与肝癌患者性别、年龄、血清甲胎蛋白(α-fetoprotein,AFP)水平、乙肝表面抗原(hepatitis B surface antigen,HBsAg)、肿瘤大小、病理学分级、静脉浸润以及是否伴随肝硬化等的相关性,结果表明血清AFP水平与GPC3表达呈正相关(P=0.007),HBsAg与DKK-1表达呈正相关(P=0.037),DKK-1阳性染色与肝细胞癌组织分级存在相关性(P=0.014),与其他参数则无相关性。结论:GPC3在肝细胞癌组织的阳性染色率为47.37%,GPC3染色阴性的肝细胞癌组织中DKK-1和DKK-2双阳性染色率为40.00%,而GPC3(+)联合DKK-1(+)/DKK-2(+)/GPC3(-)可将肝细胞癌的免疫组织化学检出率提高至68.42%(39/57),降低肝癌免疫组织化学检测中的假阴性率。展开更多
AIM To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan(HSPG), clone 1E4-1D9.METHODS The antigen recognized by...AIM To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan(HSPG), clone 1E4-1D9.METHODS The antigen recognized by m Ab 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3(https://www.ebi.ac.uk/Tools/msa/clustalo/). The competitive reaction of mA b 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mA b 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mA b 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry.RESULTS Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by m Ab 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350(1478 amino acid) and gi60219551(1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mA b 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of m Ab 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mA b 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by m Ab 1E4-1D9 was studied. The results demonstrated that m Ab 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines. CONCLUSION All findings show that mA b 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies.展开更多
文摘目的:探讨DKK-1、DKK-2和人磷脂酰肌醇蛋白聚糖3(glypican3,GPC3)蛋白在肝细胞癌组织中的表达及临床意义。方法:采用组织芯片联合免疫组织化学法检测DKK-1、DKK-2和GPC3蛋白在10例正常肝、12例肝硬化、57例肝细胞癌及癌旁肝组织中的表达差异,并分析其临床意义。结果:免疫组织化学检测结果发现,DKK-1和DKK-2在肝细胞癌组织中阳性染色率分别为59.65%(34/57)和57.89%(33/57);GPC3只在肝细胞癌组织中表达,其阳性染色率为47.37%(27/57),而在正常肝、肝硬化及癌旁肝组织中均呈阴性表达。DKK-1与DKK-2阳性表达之间存在密切相关性(χ2=0.570,P=0.000),DKK-2与GPC3阳性染色之间也存在相关性(χ2=0.272,P=0.041)。统计分析DKK-1、DKK-2及GPC3在肝细胞癌组织中阳性染色与肝癌患者性别、年龄、血清甲胎蛋白(α-fetoprotein,AFP)水平、乙肝表面抗原(hepatitis B surface antigen,HBsAg)、肿瘤大小、病理学分级、静脉浸润以及是否伴随肝硬化等的相关性,结果表明血清AFP水平与GPC3表达呈正相关(P=0.007),HBsAg与DKK-1表达呈正相关(P=0.037),DKK-1阳性染色与肝细胞癌组织分级存在相关性(P=0.014),与其他参数则无相关性。结论:GPC3在肝细胞癌组织的阳性染色率为47.37%,GPC3染色阴性的肝细胞癌组织中DKK-1和DKK-2双阳性染色率为40.00%,而GPC3(+)联合DKK-1(+)/DKK-2(+)/GPC3(-)可将肝细胞癌的免疫组织化学检出率提高至68.42%(39/57),降低肝癌免疫组织化学检测中的假阴性率。
基金Supported by National Research Council of Thailand(NRCT),No.2559A10402115
文摘AIM To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan(HSPG), clone 1E4-1D9.METHODS The antigen recognized by m Ab 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3(https://www.ebi.ac.uk/Tools/msa/clustalo/). The competitive reaction of mA b 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mA b 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mA b 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry.RESULTS Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by m Ab 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350(1478 amino acid) and gi60219551(1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mA b 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of m Ab 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mA b 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by m Ab 1E4-1D9 was studied. The results demonstrated that m Ab 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines. CONCLUSION All findings show that mA b 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies.
