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目的探讨胆红素对正常人肝细胞系L-02生长及凋亡的影响。方法将不同浓度的胆红素(终浓度分别为0、100、150、200、250、300 mg/L)作用于体外培养的正常人肝细胞L-02,分别通过光镜、Hoechst 33258荧光染色、四甲基偶氮唑蓝(MTT)法、DNA琼脂糖凝胶电泳、流式细胞仪Annexin-V和PI双标等技术观察不同浓度胆红素对L-02细胞的相对存活率的影响、细胞的凋亡梯度和凋亡率。结果经不同浓度胆红素溶液作用后,L-02细胞的增殖能力明显下降,其作用呈剂量依赖性;光镜观察显示凋亡细胞变圆,体积缩小;Hoechst 33258荧光强染;MTT法发现细胞生长受到抑制;经琼脂糖凝胶电泳发现200、250、300 mg/L胆红素溶液组可见到DNA梯形条带;流式细胞仪检测示各实验组细胞凋亡率为(1.24±0.72)%、(5.88±1.66)%、(19.69±3.52)%、(31.11±6.94)%、(57.3±8.71)%;均明显高于对照组细胞凋亡率(1.16±0.58)%(P<0.01)。结论胆红素可抑制L-02细胞生长并诱导其凋亡。
Objective To investigate the effects of bilirubin on the growth and apoptosis of normal human hepatocyte cell line L-02. Methods Different concentrations of bilirubin (final concentrations of 0, 100, 150, 200, 250 and 300 mg / L) were respectively cultured on normal human hepatocytes L-02 in vitro and were observed by light microscopy, Hoechst 33258 fluorescence staining , MTT assay, DNA agarose gel electrophoresis, flow cytometry Annexin-V and PI double labeling were used to observe the effect of different concentrations of bilirubin on the relative survival rate of L-02 cells , Cell apoptosis gradient and apoptosis rate. Results The proliferative ability of L-02 cells was significantly decreased by different concentrations of bilirubin solution in a dose-dependent manner. The apoptotic cells were round and reduced in volume by fluorescence microscopy. Hoechst 33258 fluorescence staining and MTT assay Cell growth was inhibited. DNA ladder banding was observed in 200, 250 and 300 mg / L bilirubin solution by agarose gel electrophoresis. Flow cytometry showed that the apoptotic rates in each experimental group were (1.24 ± 0.72 ), (5.88 ± 1.66)%, (19.69 ± 3.52)%, (31.11 ± 6.94)% and (57.3 ± 8.71)%, respectively, were significantly higher than those in the control group (1.16 ± 0.58)% 0.01). Conclusion Bilirubin can inhibit the growth of L-02 cells and induce their apoptosis.