论文部分内容阅读
目的:检测华中某癌症高发地区环境水样有机提取物诱导人肝细胞系L-02细胞转化的能力,并初步探讨其诱导细胞转化的表观遗传学机制。方法:分别采集研究地区河水、离河较近(约1 km)及离河较远(约20 km)的浅井水(约10 m),用固相萃取方法提取其中的有机物。用台盼蓝染色计数法和单细胞凝胶电泳试验检测受试物的细胞毒性和致DNA损伤效应,以受试物对L-02细胞进行长期染毒并利用软琼脂克隆形成试验和裸鼠皮下成瘤试验检测受试物诱导L-02细胞转化的能力。采用甲基化特异性PCR(methyrlation specific PCR,MSP)技术检测转化细胞中O~6-甲基鸟嘌呤-DNA甲基转移酶(O~6-methylguanine-DNA methyhransferase,MGMT)基因启动子区甲基化状况,并分别用实时荧光定量PCR和Western blot技术检测转化细胞MGMT mRNA和蛋白的表达水平。结果:河水、离河较近的井水、离河较远的井水的有机提取物处理L-02细胞24 h后,细胞存活率达70%的处理浓度分别为每毫升培养基30、150、200ml原水,且均含有致DNA损伤的物质。以此作为最高剂量对细胞进行长期染毒,12周以后各处理组细胞软琼脂克隆形成试验和裸鼠皮下成瘤试验均显示转化特性。MSP结果显示转化细胞中MGMT基因启动子区发生高甲基化,与DMSO溶剂对照组比较,转化细胞中MGMT mRNA和蛋白表达水平均下调(P<0.05)。结论:研究地区环境水样有机提取物具有诱导L-02细胞转化的能力,MGMT基因启动子区的高甲基化可能参与有机物诱导的细胞转化过程。
OBJECTIVE: To detect the ability of organic extracts of environmental water samples from high-risk areas in central China to induce the transformation of L-02 cell line, and to explore its epigenetic mechanism. Methods: The river water in the study area was collected, and the shallow water (about 10 m) far from the river (about 1 km) and further away from the river (about 20 km) were collected. The organic matters were extracted by solid-phase extraction. The cytotoxicity and DNA damaging effects of the test substance were detected by trypan blue staining and single cell gel electrophoresis test. L-02 cells were exposed to the test substance for a long time and the soft agar colony formation assay and nude mice Subcutaneous tumorigenicity assay tests the ability of the test substance to induce L-02 cell transformation. Methylation-specific PCR (MSP) was used to detect the promoter region of O ~ 6-methylguanine-DNA methyhransferase (MGMT) in transformed cells The expression of MGMT mRNA and protein in transformed cells was detected by real-time fluorescence quantitative PCR and Western blot respectively. Results: After treatment of L-02 cells with river water, well water near river and well water far away from the river for 24 h, the cell viability at 70% was 30,150 , 200ml of raw water, and contain DNA damage caused by substances. As the highest dose of the cells for long-term exposure, 12 weeks after the treatment group cells soft agar colony formation assay and nude mice subcutaneous tumor test showed transformation characteristics. MSP results showed that MGMT gene promoter hypermethylation occurred in transformed cells. Compared with DMSO solvent control group, the expression of MGMT mRNA and protein in transformed cells were down-regulated (P <0.05). CONCLUSION: The study of regional environmental water-based organic extract has the ability to induce L-02 cell transformation. The hypermethylation of MGMT gene promoter region may be involved in the process of organic-induced cell transformation.