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目的检测胃癌细胞线粒体基因组(mtDNA)12SrRNA的变异,探讨其与胃癌发生的关系及意义。方法采用PCR产物直接测序法检测22例胃癌组织及其对应胃癌远端正常组织的细胞线粒体12SrRNA的变异;对其中发生特异性突变的癌组织细胞和异型增生组织细胞采用激光捕获显微切割技术进行分离,采用变性高效液相色谱法(DHPLC)、等位特异性PCR、巢式PCR和聚丙烯酰胺凝胶电泳,进一步分析该突变的性质及其突变体在癌和异型增生细胞中的定量差异;RNAdraw软件分析12SrRNA突变体RNA二级结构变化。结果(1)与mitomap线粒体数据库比较后发现一些新的变异位点,其中np652G插入和np716T G颠换仅在癌组织中。(2)12SrRNA变异的频度在弥漫型胃癌(5/17,29.4%)和肠型胃癌(12/17,70.6%)间差异有统计学意义(P<0.05)。(3)线粒体12SrRNA np652G插入和716T G颠换为异质性突变,该突变体在癌组织细胞(0.89±0.02)中的相对量高于异型增生组织细胞(0.68±0.09,P<0.01)。(4)652G的插入不利于12SrRNA局部RNA二级结构的稳定,而T G颠换等则影响有限。结论(1)12SrRNA高变异率可能与肠型胃癌的发生有关;(2)大部分变异共存于胃癌和正常胃组织中,可能不具有肿瘤特异性;(3)np652G插入和np716T G突变对胃癌发生可能有一定的作用;(4)在正常胃黏膜异型增生胃癌演?
Objective To detect the variation of mitochondrial genome (mtDNA) 12SrRNA in gastric cancer cells and to explore its relationship with gastric carcinogenesis and its significance. Methods Direct sequencing of PCR products was used to detect the mitochondrial 12SrRNA variation in 22 gastric cancer tissues and their corresponding normal tissues distal to gastric cancer. Laser-capture microdissection was performed on the cancerous cells and dysplastic tissue cells with specific mutations The quantitative analysis of the nature of the mutation and its mutants in cancerous and dysplastic cells was further analyzed by denaturing high performance liquid chromatography (DHPLC), allele-specific PCR, nested PCR and polyacrylamide gel electrophoresis ; RNAdraw software analysis 12SrRNA mutant RNA secondary structure changes. Results (1) Compared with the mitomap mitochondrial database, some new mutation sites were found, in which np652G insertion and np716T G transversion were only in the cancer tissues. (2) The frequency of 12SrRNA mutation was significantly different between diffuse gastric cancer (5 / 17,29.4%) and intestinal type gastric cancer (12 / 17,70.6%) (P <0.05). (3) The relative amount of mitochondrial 12SrRNA np652G insertion and 716T G transversion to heterogeneous mutation was higher than that of dysplasia (0.68 ± 0.09, P <0.01) in cancer tissue cells (0.89 ± 0.02). (4) The insertion of 652G is not conducive to the stability of the secondary RNA secondary structure of 12SrRNA, but the effect of T G transversion is limited. Conclusions (1) The high mutation rate of 12SrRNA may be related to the occurrence of intestinal-type gastric cancer. (2) Most of the mutations co-exist in gastric cancer and normal gastric tissue and may not have tumor-specificity. (3) The np652G insertion and np716T G mutation affect gastric cancer Occurrence may have a role; (4) in gastric dysplasia of gastric dysplasia?