牛乳铁蛋白素对Jurkat细胞和HFL-I细胞生物学特性的影响

目的形态学观察牛乳铁蛋白素(LfcinB)对Jurkat细胞与人胚肺成纤维(HFL-I)细胞作用的程度,验证LfcinB诱导Jurkat细胞凋亡信号转导通路。方法用荧光显微镜观察Hoechst33258染色LfcinB处理过的Jurkat细胞和正常成纤维细胞,与阴、阳性对照组比较;用DNA断裂琼脂糖凝胶电泳分析LfcinB对Jurkat和HFL-I细胞作用差异;用流式细胞术分析Jurkat细胞早期凋亡和线粒体电势电位状况;Westernblotting研究Jurkat细胞接触LfcinB4、6和8h细胞内caspase-3、caspase-8、caspase-9和胞浆中细胞色素C含量的变化。结果在LfcinB作用Jurkat细胞DNA断裂琼脂糖凝胶电泳图中显示为阶梯状凋亡DNALadder,而LfcinB作用的HFL-I细胞无此现象;在荧光显微镜可以看到LfcinB作用过的Jurkat细胞经Hoechst33258染色细胞核呈致密浓染和HFL-I细胞的细胞核呈均匀染色;流式术分析结果为LfcinB作用Jurkat细胞2、4h的凋亡率分别为11.5%和17.8%,线粒体膜电位下降;caspase-3、caspase-9和细胞色素C的免疫印记分析显示,LfcinB能使Jurkat细胞胞浆中的细胞色素C含量增加,被激活caspase-3、caspase-9在胞浆中逐渐增多,对caspase-8没有影响。结论LfcinB诱导Jurkat细胞凋亡,对正常成纤维细胞没有作用;Jurkat细胞接触LfcinB2h以后细胞内caspase-3、caspase-9和胞浆中细胞色素C的含量累积增加,验证了LfcinB通过依赖caspase家族的细胞内信号通路诱导Jurkat细胞凋亡。

IMPACT OF BOVINE LACTOFERRICIN ON BIOLOGICAL FEATURES IN JURKAT T LEUKEMIA CELLS AND HFL-I CELLS

Objective To study the morphology and the effect of bovine lactoferricin (LfcinB) for Jurkat T leukemia cells and HFL-I cell and further validate and analyze the signal transduction passage way of LfcinB-induced apoptosis. Method The Jurkat T leukemia cells and fibroblasts stained with Hoechst 33258 after in vitro treatment with LfcinB were observed through fluorescence microscope, compared with positive and negative control groups to analyze the difference between the effect of LfcinB on Jurkat T leukemia cells and HFL-I cell with gel electrophoresis analysis of DNA fragmentation. Early apoptosis and mitochondrial membrane potential of Jurkat T leukemia cells were analyzed by flow cytometry, and the changes of caspase-3, caspase-8, caspase-9 and cytochrome C in endochylema of Jurkat T leukemia cells after exposed to LfcinB during 4, 6 and 8 h were detected by Western blotting. Results DNA Ladder was shown by gel electrophoresis analysis of DNA fragmentation in Jurkat T leukemia cells following treatment with LfcinB, which was not found in HFL-I cell. Through fluorescence microscope, we found that nucleus of LfcinB-exposed Jurkat T leukemia cells stained with Hoechst 3325 became condense, but the nucleus of HFL-I cell was not changed. The results of flow cytometry analysis indicated that apoptosis rates of Jurkat T leukemia cells exposed to LfcinB for 2, 4 h were 11.5% and 17.8% respectively, with decline of mitochondria membrane potential. Immunoblot analysis showed that LfcinB could increase the content of cytochrome C, with the activated caspase-3 and caspase-9 increase gradually in endochylema of Jurkat T leukemia cells, however there was no effect of LfcinB on caspase-8. Conclusion LfcinB induced apoptosis of Jurkat T lenkemia cells, but not affected fibroblasts. After Jurkat T leukemia cells, contacted with LfcinB for more than 2 h, the contents of caspase-3, caspase-9 and cytochrome C in the cells were cumulatively increasing, which further validated that LfcinB induced Jurkat apoptosis by intra-cellular signal pathway depending on caspase family.