常见结直肠癌无线粒体DNA细胞株的建立

The Establishment of Common Colorectal Cancer Cell Lines with Mitochondrial DNA-depleted

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： Higher mitochondrial DNA （mtDNA） content was significantly associated with an elevated risk and poor prognosis of colorectal cancer cell However, its effect on cell and the mechanism is unclear due to lack of appropriate cellular models. Therefore it is important to establish colorectal cancer rho 0 （ρ 0） cell lines （mitochondrial DNA-depleted）. Methods： Colorectal cancer cell lines SW480, HCT 116, HCT-8 were cultured in 1640 medium with 10% FBS, pyruvate, uridine and different concentrations of ethidium bromide. The changes of mtDNA content were detected by real-time quantitative PCR. The changes of the mitochondrial number and morphology between ρ 0 cells and control cells were examined under microscope. Results： SW4800 0 cell line can be obtained by treating SW480 with 50 ng/mL EB for 25 passages and then with 100 ng/mL EB for 14 passages; HCT 116ρ 0 cell line can be gotten by treating HCT116 cells with 100 ng/mL EB for 32 passages, 200 ng/mL EB for 10 passages and 500 ng/mL EB for 3 passages in orderly; HCT-8ρ 0 ceils can be harvested by treating HCT-8 cells with 200 ng/mL EB for 24 passages. Furthermore, ρ 0 cells become larger and longer compared to their parental cells. The mitochondria mass in p 0 cells increases and the shape of mitochondria becomes larger and longer. Conclusions：0 0 cells can be successfully obtained by being treated with EB. But there are still differences between different colorectal cancer cell lines. Furthermore reducing mtDNA copy numbers can significantly influence the shape of colorectal cancer cells.