人肝癌细胞线粒体DNA变异研究

Mitochondrial DNA alterations analysis in human hepatocellular carcinoma cells

目的比较肝癌细胞中线粒体DNA变异，探讨线粒体DNA变异与肝癌细胞成瘤能力、血清饥饿耐受性以及对化疗药物敏感性的关系。方法DNA直接测序法检测肝癌细胞中线粒体DNA变异；噻唑蓝（MTT）比色法检测酐癌细胞活力和顺铂对肝癌细胞生长抑制率，裸鼠皮下接种肝癌细胞检测成瘤能力。结果肝癌细胞线粒体D—loop区变异点数目：肝星形细胞（HSC，16）、BEL-7402（11）、HepG2（11）；环氧化酶Ⅱ（COXII）基因变异点数目：HSC（2）、BEL-7402（1）、HepG2（3），另外HepG2还存在8个碱基的片段缺失；HSC与HepG2细胞中线粒体DNA拷贝水平较低，BEL-7402较高。HSC和BEL-7402细胞活力明显大于HepG2；细胞接种23天后，HSC成瘤能力最强，其次是BEL-7402；HepG2未见成瘤。HSC对血清饥饿的耐受力最强，其次是HepG2，BEL-7402较弱；10μmol顺铂作用72h对BEL-7402抑制作用最强，其次是HepG2和HSC。结论肝癌干细胞中D．100p区突变点数目较肝癌细胞明显增多；HepG2细胞成瘤能力低可能与COXⅡ基因严重变异有关；肝癌细胞线粒体拷贝数与其对血清饥饿耐受性呈负相关，对顺铂敏感性呈正相关。

Objective To compare mitochondrial DNA alterations in hepatocellular carcinoma cells and the relationship between mitochondrial DNA alterations and tumorigenesis, serum starvation tolerance and chemotherapy sensitivity. Methods Direct DNA sequencing methods were used to detect the variants of the mtDNA in human hepatocellular carcinoma cells. Cell viability and cell growth inhibition rate of cisplatin were tested by methyl thiazol tetrazolium （MTY） . Tumorigenesis was examined by subcutaneously inoculating hepatocellular carcinoma cells in nude mice. Results Numbers of single variant in the mitochondrial DNA D-Loop region were： hepatic stellate cells （ HSC, 16）, BEL-7402 （ 11 ）, HepG2 （ 11 ） Numbers of single variant in the COX 1] gene were ： HSC （2）, BEL-7402 （ 1 ）, HepG2 （ 3 ） ; a 8-bp fragment deletion were detected in COX ][ gene of HepG2 cells. The level of mtDNA was significantly de- creased in HSC and HepG2 cells as compared to BEL-7402 cells. Cell viability was significandy higher in HSC and BEL-7402 cells as compared to HepG2 cells. The serum starvation tolerance ability was significantly decreased in BEL-7402 ceils as compared to HepG2 and HSC cells. The cell growth inhibition rate of cisplatin was significantly increased in BEL-7402 cells as compared to HepG2 and HSC ceils. Higher tu- morigenesis ability was detected in HSCland BEL-7402 cells, no tumorigenesis was observed in HepG2 cells after subcutaneously inoculated in n^de~ mice for 23 days. Conclusion Taken these results together, we suggest that serious variations in COX/I] gene may contribute to HepG2 cells＇ low tumorigenesis ability. There is a negative correlation between mitochondrial DNA copy number and serum starvation ability, addi- tionally; there is positive correlation between mitochondrial DNA copy number and sensitivity to cisplatin of hepatocellular carcinoma cells.