超氧阴离子自由基促肝卵圆细胞株WB-F344增殖、转化作用的研究

Study on the Role of Superoxide Anion in Promoting Proliferation and Transformation of Rat Liver Oval Cell Line WB-F344

目的 探讨超氧阴离子自由基 (O- ·2 )对肝卵圆细胞株 WB- F34 4的促增殖及转化作用。方法 用黄嘌呤 -黄嘌呤氧化酶 (X- XO)系统产生的 O- ·2 直接作用于培养的 WB- F34 4细胞 ,以 MTT比色试验、3H- Tdr掺入液闪计数、3H- Tdr掺入放射自显影观察其对细胞的促增殖效应 ;细胞经 N-甲基 - N′-硝基 - N-亚硝基胍 (MNNG)启动后 ,用小剂量的 O- ·2 (10 0μmol/L X,0 .2 m U /m l XO)连续作用促其转化 ,以形态学观察、核型分析及甲基纤维素半固体培养进行检测。结果 小剂量的 O- ·2 1次作用具有明显的促进 WB细胞增殖效应 ,连续作用 15 d,促进经 MNNG启动的 WB细胞的转化 ,形态上呈现典型的转化细胞的特征 ;核型分析显示染色体数目改变 ,染色体结构畸变率明显升高 ;在甲基纤维素半固体培养基中可形成克隆。结论 小剂量的 O- ·2 可诱使肝卵圆细胞增殖、转化 ,提示 O- ·2 在肝癌的发生中具有重要作用 。

Objective To study the role of superoxide anion (O-· 2 ) in promoting proliferation and transformation of rat liver oval cell strain WBF344. Methods WBF344 cells cultured were stimulated directly by O -· 2 generated by interaction of xanthine with xanthine oxidase (XXO). The effect of O -· 2 in promoting proliferation of WB cells was investigated by using MTT colorimetric analysis, 3 HTdr incorporation liquid scintillation counter and 3 HTdr incorporation autoradiography. WB cells initiated with NmethylN′nitroNnitrosoguanidine(MNNG) were promoted by stimulating continuously with O -· 2 of low concentration (X:100 μmol/L,XO: 02 mU/ml). The transformation effect was tested by morphologic observation, karyotype analysis and anchorageindependent growth assay. Results Proliferation of WB cells was induced obviously by O-· 2 of low concentration for only one time. WB cells initiated with MNNG were transformed by action with O -· 2 of low concentration continuously for 15 d and typical morphologic character of transformed cells was observed. In karyotype analysis the cells chromosome number changed and the frequency of structure aberration raised dramatically. Also the transformed cells could form clone on selfsolid culture medium. Conclusions The biological effect of O -· 2 was related closely with its dose; The effects of low concentration in promoting proliferation and transformation of liver oval cells indicate its important role in hepatocarcinogenesis and antioxidation was able to provide a new clue in prevention and cure of hepatoma.