构建体外错配修复功能分析模型及其在人类结直肠癌细胞株及肿瘤组织中的应用

Construction of a Model Used for the Mismatch Repair Functional Analysis in vitro and Being Used in the Human Colorectal Cancer Cell Line and Tumor Tissue

目的 构建体外错配修复 (mismatchrepair,MMR)功能分析模型 ,应用于人类结直肠癌细胞株及肿瘤组织 ,分析其整体的MMR功能状态 ;并与微卫星稳定性 (microsatellitestability ,MSS)检测结果比较 ,评价这两种检测方法在结直肠癌基础研究和临床诊断治疗中的价值。方法 以噬菌体M 13mp2为材料 ,以lacZα为报告基因 ,构建含有单碱基错配及双碱基缺失的异源双链DNA分子 ;以其为待修复模板和细胞株TK6 (作为MMR功能完整表型 )共同组成体外MMR功能分析模型。提取结肠癌细胞株Lovo及 1例遗传性非腺瘤病性结直肠癌(hereditarynon polyposiscolorectalcancer ,HNPCC) ,1例散发性直肠癌 (sporadicrectalcancer,SRC)肿瘤组织的总蛋白 ,经大T抗原依赖性SV 4 0DNA复制证实其生物学活性保持良好后与构建成功的异源双链DNA共同作用 ,通过作用前后噬斑的变化 ,计算修复效率 ,从而了解其错配修复系统功能状态。采用国际HNPCC合作小组推荐的 5个位点分析细胞及病人正常及肿瘤组织的微卫星稳定性。结果 TK6细胞株微卫星稳定 ,对双碱基缺失del(2 )的修复效率超过 6 0 % ,对单碱基错配G·G的修复效率超过 5 0 % ;而Lovo细胞株微卫星不稳 ,对双碱基缺失del(2 )的修复效率低于 2 0 % ,对单碱基错配G·G的修复效率低于 10 %。 1?

Purpose: A model for the analysis of mismatch repair (MMR) function has been constructed to measure the mismatch repair function of human colorectal cancer cell and tumor tissue. The results have been compared with the results of microsatellite stability (MSS) assay. Evaluate the values of these two measurements in the basic research and clinical diagnosis and treatment of colorectal cancer. Methods: With the bacteriophage M13mp2 and its three derivates two heteroduplex DNA molecular has been constructed one with a single base mismatch, the other with two bases deletion. The heteroduplex DNA used as the template and TK6-a lymphoblastoid B-cell line with MMR competent ability make up of the model to analyze the MMR ability of all kinds of samples in vitro. Extract from Lovo-a human colorectal cancer cell line with hMSH2 homologous deletions in the exons 3 to 8 and tumor tissues of 1 case of HNPOC (hereditary non-polyposis colorectal cancer) and 1 cases of SRC (sporadic rectal cancer), which has been measurd by large T antigen dependent SV-40 DNA replication assay, has been incubated with the heteroduplex DNA. From the change of the percentage of the mixture plaques the functional status of MMR system has been evaluated. Five sites recommended by the international co-operation group of HNPCC have been measured to analyze the microsatellites in all the cell lines, tumor tissues and normal tissues of the patients. Results: The repair efficiency of TK6 to del(2) was more than 60%, to G&middotG was more than 50%. Ther Lovo efficiency was to del(2) less than 10%, G&middotG is less than 20%. The tumor tissue of a case of NHPCC was lack of MMR ability and a case of spordic rectal cancer (SRC) still maintained MMR proficiency. Both tumor and normal tissue of the HNPCC patients and SRC patient had been measured by the microsatellits assay. The HNPCC was MSI-H and the SRC was MSS. Conclusions: The whole MMR functional status of cell line and tissue can be measured by the model used for MMR functional analysis in vitro, which is sensitive and dependable. It's very useful to be used in the patients whose family history is not clear. But its operation is oomplex and need longer time to complete. The results of the functional analysis and microsatellite assay are concordance, which indicates the recommended sites used in the HNPCC are sensitive too. The microsatellite assay is stable and quick, which is suitable in the clinical diagnosis and treatment. It's meaningful to use these two measurements together in the mechanism research and classification of the colorectal cancer and in the clinical diagnosis and treatment of it.