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目的探讨结直肠癌(colorectal cancer,CRC)线粒体DNA(mitochondrial DNA,mtDNA)拷贝数异常、线粒体DNA 4 977 bp大片段缺失与TNM分期和分化程度的关系。方法选取118例石蜡标本大肠癌组织,分别提取总DNA。以ND1和β-actin为目的基因,进行SYBR Green荧光定量PCR扩增,并用PCR扩增方法检测4 977片段缺失情况,探讨它们与不同TNM分期和分化程度之间的关系。结果 CRCⅠ和Ⅱ期癌组织平均拷贝数(2ND1/β-actin)分别为128.42±31.25和115.12±47.15,Ⅲ和Ⅳ期癌组织平均拷贝数为105.22±16.35和99.45±28.46。Ⅰ和Ⅱ期的拷贝数明显高于Ⅲ和Ⅳ期的拷贝数(P<0.01),癌组织低、中、高分化的平均拷贝数分别为101.34±41.35、112.33±42.32和127.22±31.23(P<0.01),4 977片段缺失率为15.25%(18/118),线粒体DNA4977bp缺失与患者的分期呈负相关,与分化程度无明显关系。结论线粒体DNA 4 977 bp缺失在大肠癌的早期阶段起到了重要作用,随着肿瘤的进展,拷贝数逐渐减少,线粒体DNA拷贝数的变化及大片段缺失在肿瘤的发病机制中可能起一定作用。
Objective To investigate the relationship between copy number abnormality of mitochondrial DNA (mtDNA) in colorectal cancer (CRC), deletion of 4 977 bp large fragment of mitochondrial DNA and TNM staging and differentiation. Methods 118 cases of paraffin specimens of colorectal cancer tissue were selected, total DNA was extracted. The target genes of ND1 and β-actin were amplified by SYBR Green quantitative PCR, and the deletion of 4 977 fragment was detected by PCR amplification. The relationship between them and the TNM staging and differentiation was explored. Results The average copy number (2ND1 / β-actin) of CRC Ⅰ and Ⅱ stage carcinoma tissues were 128.42 ± 31.25 and 115.12 ± 47.15, respectively. The average copy numbers of stage Ⅲ and Ⅳ carcinoma tissues were 105.22 ± 16.35 and 99.45 ± 28.46. The copy numbers of stage Ⅰ and stage Ⅱ were significantly higher than those of stage Ⅲ and stage Ⅳ (P <0.01). The average copy numbers of low, moderate and high differentiated cancer tissues were 101.34 ± 41.35, 112.33 ± 42.32 and 127.22 ± 31.23, respectively <0.01). The deletion rate of 4 977 fragment was 15.25% (18/118). The deletion of 4977 bp in mitochondrial DNA was negatively correlated with the stage of the patients, but not with the degree of differentiation. Conclusion The deletion of 4977 bp in mitochondrial DNA plays an important role in the early stage of colorectal cancer. As the tumor progresses, the number of copies gradually decreases. Changes in copy number of mitochondrial DNA and deletion of large fragments may play a role in the pathogenesis of colorectal cancer.