Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE...Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.展开更多
Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. T...Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. The most current researches associated with these genes focus on the tumor vaccination using these Ags. Few reports are concerning these genes' functions. In this study, we investigated the role of MAGE-A1 gene on NIH3T3 cells after transferring with it. Methods: Clone the MAGE-A1 into the plasmids pEGFP-C3 and pcDNA3.1, then transfer the reconstructed plasmids and primary plasmids into the NIH3T3 cells using a new transfer reagent FuGENE 6. Selecting the positively transferred cells by G418. Identified by RT-PCR, Western blot, Immunocytochemistry, Laser Scanning Confocal Microscope and Fluoroscope. The cells mobile ability was measured with Millicell-PCF. The cell cycle and apoptosis were measured with Flow Cytometry. Results:The apoptosis rate of NIH3T3 cells that transferred with control plasmid pcDNA3.1 was 13.4% and the ratios that stay in S phase and G2-M phase were 5.68% and 1.04% respectively. The apoptosis rate of NIH3T3 cells that transferred with pcDNA3.1-A1 was 0.90% and the ratios that stayed in S phase and G2-M phase were 19.31% and 13.47% respectively. The apoptosis rate of the cells that transferred with control plasmid pEGFP-C3 was 1.87%, a little higher than 1.47% of those transferred with pEGFP-C3-A1. Conclusion:The MAGE-A1 gene may enhance the cell cycle, inhibit the apoptosis and raise the mobile (ability) of NIH3T3 cells.展开更多
黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞...黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。展开更多
目的:研究肿瘤相关抗原,黑色素瘤抗原家族A成员3(melanoma antigen family A,3;MAGE-A3),在人类膀胱癌干细胞中的表达情况,并探讨其意义。方法:采用反转录聚合酶链反应(RT-PCR)技术和Western blot技术检测MAGE-A3在人类膀胱癌细胞系T24...目的:研究肿瘤相关抗原,黑色素瘤抗原家族A成员3(melanoma antigen family A,3;MAGE-A3),在人类膀胱癌干细胞中的表达情况,并探讨其意义。方法:采用反转录聚合酶链反应(RT-PCR)技术和Western blot技术检测MAGE-A3在人类膀胱癌细胞系T24细胞中及从其中分离出来的具有干细胞特性的T24侧群细胞中的表达情况;采用免疫荧光双标记技术检测MAGE-A3和膀胱癌干细胞的一个标志物CD133在T24侧群细胞中的共表达情况。结果:在mRNA和蛋白水平,MAGE-A3在具有癌干细胞特性的T24侧群细胞中的表达水平明显高于对应的母系T24细胞;MAGE-A3和膀胱癌干细胞标志物CD133在T24侧群细胞中有阳性共表达。结论:MAGE-A3在人类膀胱癌干细胞中有特异性的高表达,有望成为膀胱癌干细胞一个新的、特异性的标志物,及针对膀胱癌干细胞进行免疫治疗的一个新的靶点。展开更多
目的检测人脑膜瘤组织中黑色素瘤相关抗原MAGE-D4 m RNA的表达并分析其临床意义。方法采用逆转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)方法,检测36例脑膜瘤组织和8例正常人脑组织中MAGE-D4 m RNA的表...目的检测人脑膜瘤组织中黑色素瘤相关抗原MAGE-D4 m RNA的表达并分析其临床意义。方法采用逆转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)方法,检测36例脑膜瘤组织和8例正常人脑组织中MAGE-D4 m RNA的表达,分析MAGE-D4 m RNA的表达与脑膜瘤患者临床病理特征的关系。结果 MAGE-D4 m RNA在脑膜瘤组织中的阳性表达率为58.33%(21/36),明显高于正常人脑组织中的阳性表达率12.50%(1/8)(P<0.05);脑膜瘤组织中MAGE-D4 m RNA的表达与性别、年龄、肿瘤大小和病理类型等临床病理特征无关(P>0.05)。结论在脑膜瘤组织中MAGE-D4的表达率较高,提示MAGE-D4可能成为脑膜瘤免疫治疗的靶抗原。展开更多
文摘Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.
文摘目的:探讨黑素瘤抗原(melanoma antigen gene,MAGE)-A11对雌激素受体(estrogen receptor,ER)介导的乳腺癌MCF-7细胞增殖的影响。方法:利用RT-PCR及Western blotting筛选出ER表达阳性的人乳腺癌MCF-7细胞作为模式细胞,采用基因转染、RT-PCR和Western blotting检测MAGE-A11对17β-雌二醇(17β-E)诱导的ER下游靶基因Efp表达的影响,采用免疫共沉淀法检测MCF-7细胞中MAGE-A11和ER蛋白的相互作用,采用MTT法和克隆形成实验分别检测MAGE-A11及17β-E处理对MCF-7细胞生存率和细胞克隆形成数的影响。结果:ER阳性MCF-7细胞经17β-E处理24 h后,下游靶基因Efp的mRNA(2.97±0.16 vs 1.71±0.09,P<0.05)和蛋白表达水平显著升高(2.65±0.12 vs 0.92±0.06,P<0.05);转染MAGE-A11的MCF-7细胞经17β-E 24 h处理后,其Efp的mRNA(4.01±0.19 vs 2.97±0.16,P<0.05)及蛋白表达(3.52±0.15 vs 2.65±0.12,P<0.05)更显著增加。免疫共沉淀结果显示,外源性MAGE-A11与ER之间存在相互作用。MCF-7细胞经17β-E处理后细胞增殖率显著增加[(152±6.7)%vs(108±4.8%),P<0.05],转染MAGE-A11的MCF-7细胞经17β-E处理后细胞增殖率更显著增加[(181±8.6)%vs(152±6.7)%,P<0.05];17β-E处理后MCF-7细胞克隆形成数显著增多[(77±5)vs(18±2)个,P<0.05],转染MAGE-A11的MCF-7细胞经17β-E处理后细胞的克隆形成数更显著增加[(125±6)vs(77±5)个,P<0.05)。结论:在ER阳性的乳腺癌MCF-7细胞中,MAGE-A11可通过与ER的相互作用增强ER介导的Efp的表达,从而促进细胞增殖,MAGE-A11可能成为ER阳性乳腺癌内分泌治疗耐药的靶基因。
文摘Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. The most current researches associated with these genes focus on the tumor vaccination using these Ags. Few reports are concerning these genes' functions. In this study, we investigated the role of MAGE-A1 gene on NIH3T3 cells after transferring with it. Methods: Clone the MAGE-A1 into the plasmids pEGFP-C3 and pcDNA3.1, then transfer the reconstructed plasmids and primary plasmids into the NIH3T3 cells using a new transfer reagent FuGENE 6. Selecting the positively transferred cells by G418. Identified by RT-PCR, Western blot, Immunocytochemistry, Laser Scanning Confocal Microscope and Fluoroscope. The cells mobile ability was measured with Millicell-PCF. The cell cycle and apoptosis were measured with Flow Cytometry. Results:The apoptosis rate of NIH3T3 cells that transferred with control plasmid pcDNA3.1 was 13.4% and the ratios that stay in S phase and G2-M phase were 5.68% and 1.04% respectively. The apoptosis rate of NIH3T3 cells that transferred with pcDNA3.1-A1 was 0.90% and the ratios that stayed in S phase and G2-M phase were 19.31% and 13.47% respectively. The apoptosis rate of the cells that transferred with control plasmid pEGFP-C3 was 1.87%, a little higher than 1.47% of those transferred with pEGFP-C3-A1. Conclusion:The MAGE-A1 gene may enhance the cell cycle, inhibit the apoptosis and raise the mobile (ability) of NIH3T3 cells.
文摘黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。
文摘目的:研究肿瘤相关抗原,黑色素瘤抗原家族A成员3(melanoma antigen family A,3;MAGE-A3),在人类膀胱癌干细胞中的表达情况,并探讨其意义。方法:采用反转录聚合酶链反应(RT-PCR)技术和Western blot技术检测MAGE-A3在人类膀胱癌细胞系T24细胞中及从其中分离出来的具有干细胞特性的T24侧群细胞中的表达情况;采用免疫荧光双标记技术检测MAGE-A3和膀胱癌干细胞的一个标志物CD133在T24侧群细胞中的共表达情况。结果:在mRNA和蛋白水平,MAGE-A3在具有癌干细胞特性的T24侧群细胞中的表达水平明显高于对应的母系T24细胞;MAGE-A3和膀胱癌干细胞标志物CD133在T24侧群细胞中有阳性共表达。结论:MAGE-A3在人类膀胱癌干细胞中有特异性的高表达,有望成为膀胱癌干细胞一个新的、特异性的标志物,及针对膀胱癌干细胞进行免疫治疗的一个新的靶点。
文摘目的检测人脑膜瘤组织中黑色素瘤相关抗原MAGE-D4 m RNA的表达并分析其临床意义。方法采用逆转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)方法,检测36例脑膜瘤组织和8例正常人脑组织中MAGE-D4 m RNA的表达,分析MAGE-D4 m RNA的表达与脑膜瘤患者临床病理特征的关系。结果 MAGE-D4 m RNA在脑膜瘤组织中的阳性表达率为58.33%(21/36),明显高于正常人脑组织中的阳性表达率12.50%(1/8)(P<0.05);脑膜瘤组织中MAGE-D4 m RNA的表达与性别、年龄、肿瘤大小和病理类型等临床病理特征无关(P>0.05)。结论在脑膜瘤组织中MAGE-D4的表达率较高,提示MAGE-D4可能成为脑膜瘤免疫治疗的靶抗原。
