【作者】 罗彬；

【导师】 谢小薰；

【作者基本信息】 广西医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 我们前期的研究发现癌-睾丸抗原OY-TES-1在肝癌中高表达,在正常组织中限制性表达,并且能够引起机体的体液免疫反应,提示它是一个颇有运用开发前景的靶抗原。本研究初步研究OY-TES-1的表达对肝癌细胞的部分生物学功能的影响;了解恶性肿瘤(结直肠癌)和良性肿瘤(脑膜瘤)中OY-TES-1的表达情况及其抗体出现的情况,探讨将其应用于辅助诊断及肿瘤免疫治疗的可行性。第一部分OY-TES-1对肝癌细胞生物学行为影响的体外实验研究目的:构建OY-TES-1真核表达载体pEGFP-N1/OY-TES-1,稳定转染至肝癌细胞株HepG2,体外观察OY-TES-1对HepG2生长、增殖及迁移的的影响。方法:根据人OY-TES-1基因序列设计引物,以pMAL-C2/OY-TES-1重组质粒作为模板进行PCR,扩增出OY-TES-1编码区cDNA,并与pEGFP-N1质粒连接,构建成pEGFP-N1/OY-TES-1重组质粒,经DNA测序鉴定后,用Fugene HD将重组质粒转染至OY-TES-1表达阴性的肝癌细胞株HepG2。G418筛选阳性克隆并扩增、培养、传代,获得稳定转染株。分别用稳定表达重组质粒pEGFP-N1/OY-TES-1、空质粒pEGFP-N1及未转染的HepG2,通过细胞计数、MTT比色法、平板克隆形成、细胞周期测定和划痕修复等一系列细胞功能学实验,观察获得OY-TES-1表达后的HepG2细胞其生长、增殖和迁移改变。结果:测序及序列比对证实,pEGFP-N1/OY-TES-1真核表达载体中插入OY-TES-1的基因序列完全正确。RT-PCR和IHC结果显示:在pEGFP-N1/OY-TES-1重组质粒稳定转染的HepG2细胞中,OY-TES-1的表达较空质粒转染细胞和未转染细胞增强。对pEGFP-N1/OY-TES-1重组质粒和pEGFP-N1空质粒转染及未转染的HepG2细胞进行检测,发现OY-TES-1的转染对HepG2的生长、增殖及迁移能力无明显影响。结论:成功构建了pEGFP-N1/OY-TES-1真核表达载体,OY-TES-1对肝癌细胞株HepG2的生长、增殖及迁移无明显影响。第二部分OY-TES-1在结直肠癌和脑膜瘤的表达及临床意义分析目的:研究OY-TES-1在结直肠癌和脑膜瘤的表达及临床意义。方法:采用RT-PCR和IHC方法,分别检测24例腺瘤组织、60例结直肠癌及相应的癌旁组织、45例脑膜瘤组织OY-TES-1mRNA和蛋白质;结合临床病理资料对实验结果进行统计分析。结果: RT-PCR结果显示,结直肠癌OY-TES-1mRNA阳性率为73.3%,明显高于癌旁组织(55.0%)和腺瘤组织(45.8%)(P<0.05);脑膜瘤OY-TES-1mRNA阳性率为66.7%。免疫组织化学法检测发现,OY-TES-1蛋白在腺瘤组织和结直肠癌癌旁组织中均无表达,结直肠癌和脑膜瘤OY-TES-1蛋白阳性率分别为43.3%和40.0%。统计学分析结果显示,结直肠癌OY-TES-1蛋白的表达与浸润深度和组织分化程度相关(P<0.05),与年龄、性别、组织类型、发病部位、肿瘤大小、淋巴结转移、远处转移、TNM分期及Duke’s分期无关;脑膜瘤中OY-TES-1的表达与年龄、性别及组织类型均无关。结论:OY-TES-1蛋白较之其mRNA更具有限制性表达的特点。OY-TES-1蛋白主要在进展期结直肠癌中表达,其表达可能与肿瘤的演进和恶化有关;结直肠癌和脑膜瘤中OY-TES-1的表达均较高,而癌旁组织和癌前病变组织不表达,OY-TES-1对于肿瘤的辅助诊断及免疫治疗可能具有潜在的应用价值。第三部分结直肠癌和脑膜瘤患者血清OY-TES-1抗体的检测目的:了解结直肠癌和脑膜瘤患者血清中OY-TES-1抗体的出现情况,探讨OY-TES-1血清抗体出现的临床意义。方法:采用ELISA检测25例结肠癌和31例脑膜瘤患者血清中相应的OY-TES-1抗体,以50例正常人血清作为对照。结合临床病理资料对实验结果进行统计分析。结果:ELISA检测未发现正常人血清中有OY-TES-1抗体,结直肠癌和脑膜瘤患者血清抗体阳性率分别为8%和16.3%。OY-TES-1血清抗体的出现与临床资料无明显关系。结论:OY-TES-1在部分结直肠癌和脑膜瘤患者血清出现特异性抗体,其意义尚需进一步的探讨。更多还原

【Abstract】 Our previous study found that cancer-testis antigen OY-TES-1 was highly expressed in liver cancer, restrictively expressed in normal tissues. There was humoral immune response against OY-TES-1 in cancer patients . It is suggested that it may be a promising target antigen . This study will focuse on elucidating the some of biological function of tumor cells with OY-TES-1 expression, understaning the expression of OY-TES-1 in malignant (coloretal cancer) and benign tumor (meningioma), and humoral response in those patients. The results will help to evaluate the feasibility for immunotherapy of OY-TES-1.Part 1 In vitro experimental study of OY-TES-1 expression in cancer cell effecting on its biological functionAIM: To construct the eukaryotic expression vector pEGFP-N1/OY-TES-1 which then transfect stably into hepatocellular carcinoma cell line HepG2. Transfected cells will be observed in vitro for their growth, proliferation and migration.METHODS: Primers were designed according to the sequence of OY-TES-1 gene in human. Recombinant pMAL-C2/OY-TES-1 was used as a template for amplifing OY-TES-1 cDNA coding region. The PCR product was ligated into pEGFP-N1 vector to construct a recombinant pEGFP-N1/OY-TES-1 identified by DNA sequencing. With Fugene HD reagent, pEGFP-N1/OY-TES-1 was transfected into HepG2 cells that are OY-TES-1 negative expression. Positive clones were screened by using G418, which resulted in gain of cells with stable expression of OY-TES-1. HepG2 cells transfected with pEGFP-N1/OY-TES-1, pEGFP-N1 and untransfected cells, respectively, were tested for their function by cell counting, MTT growth test, plate clone forming, cell cycle detecting and wound-healing experiment.RESULTS: Sequencing result by blast search was confirmed that insertion of OY-TES-1 gene was correct. RT-PCR and immunohistochemistry showed that OY-TES-1 expression in both mRNA and protein lever was higher in cells transfected by pEGFP-N1/OY-TES-1 than un-transfected and transfected by pEGFP-N1 vector only. Through the tests of cell counting, MTT growth test, plate clone formatting, cell cycle detecting and wound-healing experiment, it seems that pEGFP-N1/OY-TES-1 transfected cells do not change in growth, proliferation, cell cycle and migration.CONCLUSIONS: pEGFP-N1/OY-TES-1 eukaryotic expression vector was constructed successfully. It seems that OY-TES-1 had no effect on growth, proliferation, cell cycle and migration in hepatocellular carcinoma cell line HepG2. Part 2 Expression of OY-TES-1 in colorectal cancer and meningioma with its clinical significanceAIM: To investigate the expression of OY-TES-1 expression in colorectal cancer and meningioma, and to explore its significance.METHODS: RT-PCR and immunohistochemistry were used to detect the expression level of mRNA and protein of OY-TES-1 gene, respectively, in 24 adenoma tissues, 60 colorectal cancer tissues and adjacent noncancerous tissues as well as 45 meningioma tissues. Statistic analysis was performed combining OY-TES-1expression of and patients’clinico- pathological features.RESULTS: 73.3% of OY-TES-1 was expressed in colorectal cancer tissues, which was significantly higher than that in adjacent noncancerous tissues (55.0%) and adenomas (45.8%) (P <0.05). The meningioma tissues showed 66.7% of OY-TES-1 mRNA expression. There was no OY-TES-1 protein expression in adenoma and non-cancerous tissues of adjacent colorectal cancer. The positive rate of OY-TES-1 protein expression was 43.3% and 40.0% in colorectal cancer tissues and meningioma tissues, respectively. The OY-TES-1 protein expression in colorectal cancer tissues was significantly associated with depth of invasion and degree of differentiation, and not associated with age, gender, histological type, anatomical site, tumor size, lymph node metastasis, distant metastasis, TNM stage and Duke’s stages. There were no significantly differences in OY-TES-1 expression with age, sex and histological type in meningioma.CONCLUSIONS: It is suggested that OY-TES-1 protein had a stricter expression than its mRNA. OY-TES-1 protein was mainly expressed in advanced tumors, and its expression may be related to tumor evolution and malignance. With higher OY-TES-1 expression in both colorectal cancer and meningioma and negative OY-TES-1 expression in non-cancerous tissues,OY-TES-1 may be a useful antigen for tumor auxiliary prognosis.Part 3 Seroreactivity against OY-TES-1 in colorectal cancer and meningiomaAIM: To investigate seroreactivity against OY-TES-1 in colorectal cancer and meningioma, and to explore its clinical significance.METHODS: ELISA was used to detect OY-TES-1 serum antibody in 25 colorectal cancer, 31 meningioma patients and 50 normal donors as control. OY-TES-1 serum immunoreactivity and clinical features were statistically analyzed.RESULTS: The OY-TES-1 antibody was detected in 8.0% of colorectal cancer patients’and in 16.3% of meningioma patients’sera, while no serum reactivity was detected in 50 normal donors. There were no significantly differences between OY-TES-1 serum immunoreactivity and clinical features of patients.CONCLUSIONS: Seroactivity against OY-TES-1 presents specifically in patients with colorectal cancer and meningioma. The significance of OY-TES-1 antibody in patients needs to further explore.更多还原