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目的:流行病学研究表明结直肠癌细胞线粒体DNA(mt DNA)拷贝数变异与患者预后具有显著相关性,但因缺乏相关体外细胞模型导致其细胞生物学效应及具体分子机制尚不明确。因此研究建立结直肠癌ρ0细胞株(无mt DNA)的方对该研究域具有重大意义。方法:在含有丙酮酸、尿苷及不同浓度溴化乙锭的培养基中培养大肠癌细胞株SW480、HCT 116、HCT-8,以不加溴化乙锭的细胞为对照,利用实时定量PCR技术监测不同处理组mt DNA拷贝数的变化,并观察结直肠癌ρ0细胞线粒体形态数目及细胞形态的变化。结果:SW480细胞用50 ng/m L EB处理25代后,再以100 ng/m L EB处理14代可成功构建SW480ρ0细胞株;HCT116细胞加用EB 100 ng/m L培养32代,继续用EB 200 ng/m L培养10代,再用EB 500 ng/m L培养3代,可成功获得HCT116ρ0细胞株;HCT-8细胞用200 ng/m L EB培养24代,可成功获得HCT-8ρ0细胞株。同时,ρ0细胞较亲本细胞变大变长,其线粒体的个数增多,线粒体形态变大变长。结论:利用EB处理法可成功构建大肠癌ρ0细胞株,但不同肠癌细胞方法不尽相同。mt DNA拷贝数的降低可显著影响大肠癌细胞的形态。
OBJECTIVE: Epidemiological studies have shown that copy number variation of mitochondrial DNA (mt DNA) in colorectal cancer cells has a significant correlation with the prognosis of patients. However, the lack of relevant in vitro cell model results in the cell biological effects and the specific molecular mechanisms remain unclear. Therefore, the establishment of colorectal cancer ρ0 cell line (no mt DNA) of the party of great significance in the study. Methods: Colorectal cancer cell lines SW480, HCT 116 and HCT-8 were cultured in medium containing pyruvate, uridine and different concentrations of ethidium bromide. The cells without ethidium bromide were used as control. Technique to monitor the changes of mt DNA copy number in different treatment groups and observe the change of mitochondrial morphology and cell morphology in ρ0 cells of colorectal cancer. Results: After SW480 cells were treated with 50 ng / m L EB for 25 generations, SW480 ρ0 cells could be successfully constructed with 100 ng / m L EB for 14 passages. HCT116 cells were cultured for 32 generations with EB 100 ng / m L, HCT-16p0 cell line was successfully obtained after 10 generations of EB at 200 ng / m L and 3 generations of EB at 500 ng / m L; HCT-8p0 cells were successfully cultured at 200 ng / m L EB for 24 passages Cell lines. At the same time, ρ0 cells grow larger and longer than the parental cells, the number of mitochondria increased, the morphology of mitochondria become larger and longer. Conclusion: The ρ0 cell line of colorectal cancer can be successfully constructed by EB treatment, but the methods of different colorectal cancer cells are different. The decrease of mt DNA copy number can significantly affect the morphology of colorectal cancer cells.