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OY-TES-1 expression on the biological behavior of the tumor and its antibody serological analysis

Author: LuoBin
Tutor: XieXiaoZuo
School: Guangxi Medical University
Course: Human Anatomy and Embryology
Keywords: Clone Transfection Plasmid Vector RT-PCR Immunohistochemistry ELISA Humoral immune response
CLC: R73-3
Type: PhD thesis
Year: 2010
Downloads: 89
Quote: 1
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Abstract


Our previous study found that cancer - testis antigen OY-TES-1 is highly expressed in the liver, restricted expression in normal tissues and can cause humoral immune response, suggesting that it is a quite promising target antigens use development. In this study, preliminary study OY-TES-1 expression on the part of the biological function of hepatoma cells influence; understanding of cancer (colorectal cancer) and benign tumors (meningiomas) of OY-TES-1 expression and antibodies case study will be applied to the diagnosis and tumor immunotherapy is feasible. The first part OY-TES-1 on hepatocellular carcinoma cell biological behaviors in vitro Objective: To construct OY-TES-1 eukaryotic expression vector pEGFP-N1/OY-TES-1, stable transfected into hepatoma cell line HepG2, in vitro OY-TES-1 was observed on HepG2 growth, proliferation and migration effects. Methods: human OY-TES-1 gene sequence primers pMAL-C2/OY-TES-1 recombinant plasmid as a template for PCR, amplified OY-TES-1 coding region cDNA, and connected with the pEGFP-N1 plasmid to construct pEGFP-N1/OY-TES-1 recombinant plasmid was identified by DNA sequencing, the recombinant plasmid with Fugene HD transfected into OY-TES-1 expression was negative hepatoma cell lines HepG2. Positive clones were amplified G418, cultured and passaged to obtain stable transfection strains. Stable expression of recombinant plasmids were used pEGFP-N1/OY-TES-1, empty plasmid pEGFP-N1 and non-transfected HepG2, by cell counting, MTT assay, colony formation, cell cycle and scratch repair a determination series of experiments cell function, observable OY-TES-1 expression in HepG2 cells after the growth, proliferation and migration changed. Results: Sequencing and sequence alignment confirmed, pEGFP-N1/OY-TES-1 eukaryotic expression vector inserted OY-TES-1 gene sequence is correct. RT-PCR and IHC results showed that: in pEGFP-N1/OY-TES-1 recombinant plasmid stably transfected HepG2 cells, OY-TES-1 expression in transfected cells compared to empty vector-transfected cells and not increased. And on pEGFP-N1/OY-TES-1 recombinant plasmid pEGFP-N1 empty plasmid transfected and non-transfected HepG2 cells tested and found OY-TES-1 transfection of HepG2 growth, proliferation and migration without significant affected. Conclusion: The successful construction pEGFP-N1/OY-TES-1 eukaryotic expression vector, OY-TES-1 on hepatocellular carcinoma cell line HepG2 growth, proliferation and migration of no significant impact. The second part OY-TES-1 in colorectal cancer and meningioma Expression and Clinical Significance Objective: OY-TES-1 in colorectal cancer and meningioma Expression and clinical significance. Methods: RT-PCR and IHC methods were used to detect 24 cases of adenoma, 60 cases of colorectal cancer and corresponding adjacent tissues, 45 cases of meningioma tissue OY-TES-1mRNA and protein; combination of clinical and pathological data on the experimental results for statistical analysis. Results: RT-PCR showed that colorectal OY-TES-1mRNA positive rate was 73.3%, significantly higher than the adjacent tissues (55.0%) and adenomas (45.8%) (P lt; 0.05); meningioma OY -TES-1mRNA positive rate was 66.7%. Immunohistochemical staining revealed, OY-TES-1 protein in colorectal adenoma tissues and no expression in adjacent tissues, colorectal cancer and meningioma OY-TES-1 protein were 43.3% and 40.0% . Statistical analysis showed that colorectal OY-TES-1 protein expression and depth of invasion and tissue differentiation degree (P lt; 0.05), with age, gender, histological type, disease location, tumor size, lymph node metastasis, distant Department metastasis, TNM stage, and Duke's staging; meningiomas OY-TES-1 expression and age, sex and histological types. Conclusion: OY-TES-1 protein is more restrictive than the mRNA expression characteristics. OY-TES-1 protein was mainly in advanced colorectal cancer, and its expression may be related to tumor evolution and deterioration of; colorectal cancer and meningiomas OY-TES-1 expression were higher, while the adjacent tissues and precancerous lesions did not express, OY-TES-1 in tumor diagnosis and immunotherapy may have potential applications. Part III colorectal cancer and meningioma patients OY-TES-1 antibody detection Objective: To understand colorectal cancer and meningioma serum OY-TES-1 antibodies appear, and explore OY-TES-1 serum antibodies The clinical significance. Methods: 25 cases of colon cancer by ELISA and 31 cases of meningioma serum corresponding OY-TES-1 antibody in 50 cases of normal human serum as a control. Combined with clinical and pathological data for statistical analysis of the experimental results. Results: ELISA failed to detect in normal human serum OY-TES-1 antibody in patients with colorectal cancer and meningioma serum antibody positive rate was 8% and 16.3%. OY-TES-1 serum antibodies appeared no significant relationship with clinical data. Conclusion: OY-TES-1 in some patients with colorectal cancer and meningioma specific antibodies appear, its significance still needs further study.

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