肝癌组织中线粒体DNA D-Loop区碱基变异与ROS水平

为了探讨ROS水平与突变的关系，对原发性肝癌线粒体DNA区的突变情况进行研究，同时对原发性肝癌患者组织细胞内ROS进行测定。选择20例原发性肝癌组织及其邻近的癌旁组织，用PCR方法将线粒体DNA D-Loop扩增后测序。组织内ROS的水平采用流式细胞技术测定。结果表明在20对原发性肝癌组织中存在8对mtDNA突变，突变率为40%,共发现突变位点53个，包括2个插入，11个缺失，40个点突变，其中T-C,C-T的转换占75%，4个属于微卫星结构。癌组织突变一般伴有癌旁组织突变，癌组织突变位点高于癌旁组织。发现一例标本的癌组织和癌旁组织均有大片段丢失。原发性肝癌组织内ROS水平明显高于癌旁对照( P<0.01),同时我们发现在区发生突变的患者的组织中ROS水平明显高于未发生突变的肝癌组织标本（P<0.01），发生突变的癌旁组织内ROS水平明显高于未发生突变的癌旁组织（P<0.01）。结论 (1)线粒体DNA D-Loop区是一个高度多态性和突变性的区域，在原发性肝癌中突变率较高。(2)肝癌患者组织细胞内ROS异常，提示肝癌的线粒体DNA发生的点突变及肝癌的发生可能与ROS升高有关。

Mutations in the D-Loop Region of Mitochondrial DNA and the ROS Level in the Tissue of Hepatocellular Carcinoma

To explore the relationship between ROS level and mutations in D-Loop region of mtDNA, mutations in the D-Loop region of mtDNA and the ROS level in primary hepatocarcinoma tissues were studied. We amplified the D-Loop region of mtDNA of 20 hepatocarcinomas and their adjacent tissue by PCR and then sequencing. ROS in tissue was measured by flow cytometry. mtDNA mutations were detected in 40%(8 of 20)tumor samples. 53 point mutations were detected in eight tumour samples,including two insertions,11 deletions and 40 point mutations.75% point mutations were T-C and C-T transition.There were four microsatellites among the mutations. Mutations in the adjacent tissues were always companied with mutations in tumor tissues.The mutation frequency in tumor tissues was higher than that in adjacent tissue.There was a larger unidentified deletion. The ROS level in hepatocarcinoma tissue was much higher than control (P<0.01).Meanwhile,we found the ROS level in hepatocarcinoma tissues with mutated mtDNA D-Loop was higher than that in hepatocarcinoma tissue with normal mtDNA D-Loop,and the ROS level in hepatocarcinoma adjacent tissue with mutated mtDNA D-Loop was higher than that in hepatocarcinoma adjacent tissue with normal mtDNA D-Loop. It was concluded that the D-Loop region of mitochondrial DNA was a highly polymorphoric and mutable region and the mutation rate was relatively high in patients with hepaticellular carcinoma,and the abnormal ROS level might be the point mutation in the mitochondrial DNA and hepatocarcinogenesis related to ROS.