肿瘤相关基因MAGE-n的发现及研究进展

黑色素瘤抗原-n(MAGE-n)基因是本实验室从肝癌细胞系中克隆得到的MAGE家族新成员,该基因与已报道的MAGE家族基因不同,但与MAGE-A家族基因具有同源性,与MAGE-6、MAGE-3同源性最高。我们分别检测了MAGE-n在不同组织、细胞中的表达和分布情况,结果发现:在正常肝组织、肝硬化组织及其它非肿瘤组织中未检测到MAGE-n的表达,而在肝细胞癌、胃癌等恶性肿瘤组织中表达率较高,且在其他不同肿瘤中的表达水平有差异。后续研究筛选鉴定出了HLA-A2限制性CTL表位,体内外实验均显示出良好的抗肝癌免疫效应。MAGE-n的这种表达模式和免疫特性与MAGE家族其他基因相同,提示其可作为肿瘤免疫治疗,尤其是肝癌免疫治疗的靶分子,为肝癌的免疫治疗研究提供新的策略。现就MAGE-n的研究进展作以下综述。     

两种黑色素瘤抗原基因在肝细胞癌中的表达

目的 检测黑色素瘤抗原MAGE-4和MAGE-10mRNA在中国人肝细胞癌（HC）中的表达，为用这两种基因编码蛋白作为疫苗对HCC患者进行免疫治疗提供依据。方法 用RT-PCR方法对48例HCC患者癌组织和相应癌旁肝组织、10例肝硬化组织和10例正常肝组织的MAGE-4和MAGE-10mRNA表达情况进行研究，并对RT-PCR扩增产物中目的基因片段进行cDNA序列测定。结果 48例HCC患者中，有15例（31.3%）表达MAGE-4，14例（29.2%)表达MAGE-10mRNA；而相应癌旁肝组织；腩硬化和正常肝组织均不表达,cDNA测序进一步证明，PCR产物中的目的基因片段为MAGE-4和MAGE-10cDNA序列，两种MAGE基因的表达与肿瘤大小。甲胎蛋白（α-FP）水平等临床指标无相关关系。结论 MAGE-4和MAGE-10在HCC组织中呈特异性的表达，面在癌旁非瘤性肝组织中均不表达，这使得以此抗原用于HCC患者的免疫治疗成为可能。     

黑色素瘤抗原基因在胃癌组织中表达的研究

目的：探讨黑色素瘤抗原(MAGE)基因在胃癌组织中的表达，为应用MAGE基因产物对胃癌患者进行特异性抗肿瘤免疫治疗提供理论依据。方法：用逆转录-聚合酶链反应(RT-PCR)技术检测30例胃癌组织及相应癌旁组织中MAGE-1、MAGE-2和MAGE-3基因的表达。结果：30例胃癌组织标本MAGE-1、MAGE-2和MAGE-3基因的表达率分别为46．7％、30．0％，和36．7％，有20例至少表达其中1种基因，而相应的癌旁组织均不表达MAGE。结论：MAGE基因在胃癌组织中有较高的表达率，其编码蛋白有望作为疫苗用于胃癌患者的免疫治疗。     

黑色素瘤相关抗原-A:食管癌免疫治疗新靶点

黑色素瘤相关抗原(melanoma-associated antigen,MAGE)-A亚家族是MAGE基因家族成员,属于癌睾丸抗原,包括MAGE-A 1～MAGE-A 12,定位于X染色体.MAGE-A在正常组织中几乎不表达,而在多种肿瘤组织中表达,其在食管癌细胞系和食管癌组织均有较高表达.MAGE-A基因编码的抗原肽可由食管癌细胞MHC Ⅰ分子提呈至细胞毒性T细胞,能够发挥特异性抗肿瘤活性.因此,以MAGE-A抗原为新靶点,对食管癌患者进行免疫治疗有良好的前景.     

三种MAGE基因在广西肝细胞癌中的表达

目的检测癌-睾丸抗原(CTA)基因MAGE-1、-3和-4在广西地区肝细胞癌(HCC)中的表达,探讨CTA作为疫苗对HCC治疗的可行性.方法用逆转录-聚合酶链反应(RT-PCR)法对25例广西HCC患者癌组织中MAGE-1、-3和-4基因mRNA的表达进行检测;随机选择MAGE-1、-3和-4阳性的RT-PCR产物各2例进行DNA序列测定.结果 MAGE-1和-3表达率均为40%(10/25),MAGE-4表达率为16%(4/25).DNA测序结果表明RT-PCR产物为MAGE-1、-3和-4基因.统计学分析显示MAGE-1、-3和-4基因的表达与HCC组织分化程度及临床分期无相关性(P>0.05),与HBV感染有相关性(P<0.05);此外,MAGE-1和-3的表达与肿瘤大小及AFP水平均存在相关性(P<0.05).在所检测的标本中,56%的标本至少表达上述1种MAGE,24% 的标本至少表达2种MAGE;16% 的标本表达3种MAGE.结论 MAGE-1和-3在广西HCC中有一定频率的表达,能否用于HCC的免疫治疗值得深入探讨.     

肝癌相关新基因HC6的全长cDNA克隆及分析

目的：染色体17p13.3区域内多态性位点D17S926是肝细胞肝癌（HCC）杂合性缺失热点,PAC579克隆含有D17S926位点,本实验目的在于从PAC579克隆中寻找与肝癌相关的表达序列（ESTs),对有意义的EST片段克隆其全长cDNA。方法：根据PAC579克隆中现有的9个代表新基因的 EST序列设计引物,对正常人肝cDNA文库作多聚酶链式反应（PCR）扩增检测,确定在肝组织中表达的EST,然后采用Southern杂交检测肝组织中表达的EST在27对肝癌和癌旁标本中杂合性生缺失（LOH）频率,选择缺失频率最高的EST克隆其全长cDNA,Nothern杂交验证后测序并作进一步分析。结果：PAC579现有9个代表新基因的EST中有3个在肝组织中表达,其中EST6有肝癌中LOH频率高达54．6％（6／11）,通过PACE－PCR方法,得到约1．8kb全长cDNA克隆,与Northern杂交结果一致,结构及同源性分析显示该基因是一新基因,编码一个包含134个氨基酸,分子量约为14．7kD的蛋白质,另外发现碱基序列313－592属于ALu同源序列,重新命名为肝癌相关基因6（HC6）。结论：通过定位克隆方法从肝癌杂合性缺失热点得到一个新基因HC6,该基因与肝癌的关系有待进一步研究。     

结直肠癌中黑色素瘤抗原基因mRNA的表达及意义

目的检测结直肠癌组织中黑色素瘤抗原基因MAGE-1、3和4的表达,探讨其在结直肠癌免疫治疗及作为免疫分子标志物的可行性。方法采用RT-PCR方法,检测65例结直肠癌组织和相应正常粘膜组织中MAGE-1、3和4的mRNA表达,并对PCR扩增产物进行DNA测序验证。结果65例结直肠癌组织标本中,53.8%(35/65)的患者至少表达一种MAGE基因。MAGE-1、3、4mRNA表达率分别是18.5%(12/65)、33.9%(22/65)和20%(13/65),表达任意两种或以上的MAGE基因的标本为27.7%(18/65),而相应正常粘膜组织均未检测到上述基因的表达。DNA测序结果表明RT-PCR产物确为目的基因片段。MAGE基因表达与患者的年龄(除外MAGE-1,P=0.011)、性别、CEA、肿瘤大小和位置、组织分化程度、淋巴结转移、肿瘤浸润深度及TNM分期无关(P>0.05)。结论结直肠癌中MAGE-3可作为肿瘤疫苗的备选基因和免疫学检测的分子标志物,具有作为肿瘤免疫治疗特异性靶位的潜在价值,而MAGE-1,4因表达率低限制了其应用价值。     

黑色素瘤抗原基因在胃癌组织中表达的研究

目的 :探讨黑色素瘤抗原 (MAGE)基因在胃癌组织中的表达 ,为应用MAGE基因产物对胃癌患者进行特异性抗肿瘤免疫治疗提供理论依据。方法 :用逆转录 -聚合酶链反应 (RT PCR)技术检测 30例胃癌组织及相应癌旁组织中MAGE 1、MAGE 2和MAGE 3基因的表达。结果 :30例胃癌组织标本MAGE 1、MAGE 2和MAGE 3基因的表达率分别为 4 6 7%、30 0 %和 36 7% ,有 2 0例至少表达其中 1种基因 ,而相应的癌旁组织均不表达MAGE。结论 :MAGE基因在胃癌组织中有较高的表达率 ,其编码蛋白有望作为疫苗用于胃癌患者的免疫治疗     

真核表达载体pEGFP-N1-Ubc9的构建及表达

目的:克隆人类Ubc9基因cDNA全长,构建Ubc9的真核表达载体并表达。方法:采用RT-PCR技术从人小细胞肺癌NCI-H446细胞总RNA中扩增Ubc9 cDNA基因片段,经过酶切鉴定后,克隆至pUCM-T载体,测序证实碱基序列无误后,再克隆至真核绿色荧光蛋白表达载体(pEGFP-N1)上,并转染到真核细胞,观察其在真核细胞中的表达。结果:测序证实克隆的Ubc9全长cDNA阅读框正确完整,酶切和序列测定证实Ubc9正确插入pEGFP-N1载体中,该重组载体能够在真核细胞中表达。结论:成功构建了真核表达载体pEGFP-N1-Ubc9。     

CPGL的基因结构及其与肝癌相关性的研究

目的 克隆carboxypeptidase of glutamate like(CPGL)全长cDNA，初步探讨它在肝癌发生发展中的作用。方法 通过5’-RACE方法和RT-PCR验证，确定(CPGL)全长序列，根据其全长与人类基因组的比较进而确定基因的染色体定位和其外显子、内含子；应用Western Blot方法检测CPGL在肝癌组织和癌旁组织中的表达；细胞克隆形成及生长曲线检测CPGL对肝癌细胞生长的影响。结果 CPGL全长2616bp，编码475个氨基酸的蛋白，染色体定位于18q22.3，蛋白产物定位于细胞质中，Western Blot结果发现CPGL在肝癌组织中的表达明显低于癌旁组织。将CPGL转染肝癌细胞系SMMC7721发现其具有抑制生长的作用。同源比较发现CPGL含有M20蛋白酶家族的保守功能域。结论 上述发现表明CPGL是具有潜在抑癌作用的基因。     

Expression of the MAGE gene family in human hepatocellular carcinoma

BACKGROUND: The 12 members of the MAGE gene family encode tumor specific antigens that are recognized by autologous cytotoxic T lymphocytes (CTL). The MAGE genes are expressed not only in melanoma but in the other malignant tumors as well. There is, however, little information on their expression in hepatocellular carcinoma (HCC). The authors thus studied the expression of the MAGE gene family in human HCC and discuss the possibility of specific immunotherapy using MAGE peptides. METHODS: Tumor tissue samples of HCC and paired nontumor tissues of the liver were obtained from 22 HCC patients. Total RNA was extracted and cDNA was synthesized. Polymerase chain reaction amplification using each MAGE gene specific primer was then performed to detect the expression of each MAGE gene. Immunoblotting and immunohistochemical analysis were performed to confirm the expression of MAGE-3 gene product in HCC. RESULTS: The expression rate of each MAGE gene was as follows: MAGE-1 and -3 were expressed in approximately 68% of the tumors; MAGE-8 was expressed in 46%; and MAGE-2, -6, -10, -11, and -12 were expressed in approximately 30%. Nineteen (86%) of 22 tumors expressed at least 1 MAGE gene. On the other hand, no expression was detected in the noncarcinomatous liver tissue specimens. Actual expression of the gene product of MAGE-3 was detected in 50% of tumors. Clinicopathologic data on the MAGE positive and negative cases were compared. Significant differences were observed between MAGE expression status and a few clinicopathologic factors; however, further investigation is required to elucidate these correlations completely. CONCLUSIONS: These findings demonstrated that MAGE gene expression is frequent in HCC, thus suggesting that HCC patients may be good candidates for specific immunotherapy using MAGE peptides.     

Immunohistochemical detection of MAGE tumor-associated antigens in esophageal squamous cell carcinoma

Genes of the MAGE family encode tumor-specific antigens recognized by cytotoxic T-lymphocytes in a variety of neoplasms. We investigated the protein expression of these antigens as related to the gene expression, in esophageal squamous cell carcinoma by using monoclonal antibodies recognizing MAGE gene products. Esophageal squamous cell carcinomas were found to express both MAGE-1 (4 out of 15 samples) and MAGE-3 (7 out of 15 samples) genes, by RT-PCR. Immunoblotting revealed MAGE-1 and MAGE-3 gene products in 2 and 6 out of 15 samples, respectively. Immunohistochemistry performed on 12 samples showed MAGE-1 protein expression, limited to single tumor cells, in 2 cases. MAGE-3 gene product was detectable in 7 cases: in 5 of them over 50% of neoplastic cells were positive. Considering the high percentages of tumor cells expressing MAGE-3 antigen, the use of epitope-based vaccines could be envisaged in patients displaying appropriate HLA-class I phenotype.     

MAGE, BAGE and GAGE gene expression in human rhabdomyosarcomas

MAGE, BAGE and GAGE genes encode tumor-associated antigens that are presented by HLA class I molecules and recognized by CD8(+) cytolytic T lymphocytes. These antigens are currently regarded as promising targets for active, specific tumor immunotherapy because MAGE, BAGE and GAGE genes are expressed in many human cancers of different histotype and are silent in normal tissues, with the exception of spermatogonia and placental cells. MAGE, BAGE and GAGE gene expression has been extensively studied in different tumors of adults but is largely unknown in many forms of pediatric solid cancer. Using RT-PCR, we analyzed MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, BAGE, GAGE-1,-2 or -8 and GAGE-3,-4,-5,-6 or -7b gene expression in 31 samples of pediatric rhabdomyosarcoma, the most frequent form of malignant soft tissue tumor in children. MAGE genes were expressed in a substantial proportion of patients (MAGE-1, 38%; MAGE-2, 51%; MAGE-3, 35%; MAGE-4, 22%; MAGE-6, 35%), while expression of BAGE (6%); GAGE-1, GAGE-2 and GAGE-8 (9%); and GAGE-3, GAGE-4, GAGE-5, GAGE-6 and GAGE-7B (16%) was less frequent. Overall, 58% of tumors expressed at least 1 gene, and 35% expressed 3 or more genes simultaneously. Our data suggest that a subset of rhabdomyosarcoma patients could be eligible for active, specific immunotherapy directed against MAGE, BAGE and GAGE antigens.     

黑色素瘤抗原基因MAGE-1、MAGE-3和抑癌基因p53在肺癌中表达的研究

目的:分析肺癌组织MAGE-1和MAGE-3基因以及p53基因表达及其相互关系。方法:取肺癌组织标本30例,另取癌周组织作为对照。采用逆转录聚合酶链反应(RT-PCR)方法,检测MAGE-1和MAGE-3基因以及p53基因的表达。结果:MAGE-1,MAGE-3在肺癌中有较高表达,MAGE-3的表达高于MAGE-1的表达。p53在肺癌中表达较低,在正常组织中表达较高;MAGE-1在肺腺癌中表达较鳞癌高,MAGE-3在肺鳞癌中表达较高;MAGE-1,MAGE-3的表达可能与肿瘤分化程度无关;p53在小细胞肺癌中的表达有待进一步研究;p53与MAGE在体内表达成负相关。结论:MAGE-1、MAGE-3和p53在肺癌组织与正常组织中有不同的表达,MAGE-1、MAGE-3的表达与病理类型有关,与肿瘤分化无关。     

The anti-tumor immune response induced by a combination of MAGE-3/MAGE-n-derived peptides

Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). MAGE gene products are of particular interest owing to their wide expression in many tumors and their potential to induce tumor-specific CTL responses. Antigen-specific CTLs induced by MAGE gene-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumors. MAGE-3 has been used as a target for tumor immunotherapy. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma (HCC). However, the majority of previous investigations focused on the single MAGE antigen-derived peptides as a cancer vaccine which has many limitations. The tumor antigen expression is known to be heterogeneous and tumor cells can express multiple tumor antigens. Thus, vaccines incorporating single antigen-derived epitopes may be inadequate in generating a complete immune response against the tumor. Instead, a polyvalent vaccine incorporating epitopes derived from several tumor antigens may be more effective. Our study combined the MAGE-3 and MAGE-n-derived peptides as a cancer vaccine. The results showed that the combination of MAGE-3 and MAGE-n epitopes induced more effective anti-tumor immune responses than either of the peptides alone. In addition, the peptide-specific activity was observed to be in an MHC-restricted manner. Our study indicated that the combination of several tumor antigen-derived peptides may present a better peptide-based cancer immunotherapy.