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目的探讨通过线粒体DNA(mtDNA)D-Loop区序列分析鉴别多结节性肝细胞性肝癌(肝癌)细胞克隆起源的可行性。方法实验组为多结节肝癌组,标本来自广西医科大学第一附属医院肝胆外科2004年4月至2007年8月期间连续收治的42例共112个结节性HCC行根治性手术切除的肿瘤组织。对照组来自同期单结节HCC手术切除组织共20例40个样本,分2个亚组,对照组Ⅰ为16例单结节肝癌的2块不相邻的癌组织;对照组Ⅱ为4例伴有肉眼门静脉癌栓的肝癌患者,取癌组织和癌栓各1块;正常对照组来自同期肝移植供肝或肝外伤切除的肝组织共5例。用PCR结合直接测序的方法检测各样本组织mtDNA D-Loop区的序列,并分析各例癌结节中序列异同情况。结果在42例实验组标本中,共有131个位点发生碱基变异,其中15个位点为点突变,9个位点发生插入,16个位点发生缺失,共出现98个多态性变化,位点总变异率为11.7%(131/1122,1122为mtDNAD-Loop区全长);实验组中有20例各结节的mtDNA D-Loop区序列存在差异,可能为多中心(MO)起源,22例各结节的mtDNA D-Loop区序列完全相同,可能为单中心(即肝内转移,I M)起源。对照组20例中各样本的mtDNAD-Loop区序列完全相同,为相同细胞克隆起源。正常对照组中共有14个位点发生碱基变异,均为多态性,其中NT479A>G为新的多态性。结论 mtDNA D-Loop区序列具有较高的变异率,应用PCR结合直接测序的方法检测该区序列并比较各个癌结节的DNA序列异同,可以快速、简单、有效地为区分I M和MO起源提供参考。
Objective To investigate the feasibility of identifying the origin of colony in multi-nodular hepatocellular carcinoma (HCC) by sequence analysis of D-Loop mitochondrial DNA (mtDNA). Methods The experimental group was a multi-nodular hepatocellular carcinoma (HCC) group. The specimens were obtained from 42 consecutive patients who underwent radical resection of the tumor with a total of 112 nodular HCCs from April 2004 to August 2007 in the First Affiliated Hospital of Guangxi Medical University organization. Control group from the same period of single nodules HCC surgical resection of a total of 40 cases of 40 samples, divided into 2 subgroups, control group Ⅰ 16 cases of single nodular hepatocellular carcinoma adjacent to 2 cases; control group Ⅱ 4 cases Liver cancer patients with portal vein tumor emboli in patients with cancer tissue and tumor suppository each one; normal control group from the same period of liver transplantation or liver injury resection of liver tissue in 5 cases. The sequence of mtDNA D-Loop region in each sample tissue was detected by PCR combined with direct sequencing, and the similarities and differences in the sequence of cancer nodules were analyzed. Results A total of 131 loci were found in 42 experimental groups. Fifteen loci were point mutations, nine loci were inserted, and 16 loci were deleted. There were 98 polymorphic changes , And the total site mutation rate was 11.7% (131 / 1122,1122 for mtDNAD-Loop region); in the experimental group there were 20 cases of mtDNA D-Loop region of each nodule differences, which may be multi-center (MO) The sequence of mtDNA D-Loop in each of the 22 nodules is identical, which may be the origin of single center (ie, intrahepatic metastasis, IM). The control group of 20 cases of the mtDNAD-Loop region of the same sequence, the same cell clone origin. A total of 14 loci in the normal control group were found to be polymorphic, of which NT479A> G was a new polymorphism. Conclusion The sequence of mtDNA D-Loop region has a high mutation rate. PCR and direct sequencing method can be used to detect the sequence of this region and to compare the similarities and differences of the DNA sequences of each cancer nodule, so as to provide a quick, simple and effective way to distinguish the origin of IM and MO reference.