重组人乳铁蛋白抑制口腔癌KB细胞增殖及诱导细胞凋亡

目的 研究重组人乳铁蛋白(recombinate human lactoferrin,rhLF)对人口腔KB细胞增殖及凋亡的影响，为口腔癌患者提供新的有效治疗途径提供参考。方法 采用CCK-8法检测不同浓度(0、12.5、25、50、100、200 μg/mL)的rhLF分别处理KB细胞24、48、72 h后对细胞增殖的影响；Immunofluorescence及Western blotting方法检测rhLF处理前后DNA损伤相关蛋白γ-H2AX的表达。并分别用0、50、200 μg/mL的rhLF处理KB细胞,14 d后观察其对集落克隆形成的影响；0、200 μg/mL rhLF处理KB细胞48 h后,收集细胞并使用线粒体膜电位检测试剂盒(JC-1)检测rhLF处理前后线粒体膜电位的变化。Annexin V-PI染色检测rhLF对口腔癌KB细胞凋亡的影响；Western blotting检测0、50、200 μg/mL的rhLF处理口腔癌KB细胞48 h后Parp、Caspase-3的表达情况。结果 rhLF处理KB细胞的增殖有显著的抑制作用。Immunofluorescence结果显示rhLF处理后γ-H2AX的表达明显增加，Western blotting结果也显示rhLF处理后γ-H2AX的表达成浓度依赖性增加。rhLF抑制KB细胞集落克隆的形成；JC-1荧光检测结果表明红绿荧光的相对比例降低，这种红绿荧光的相对比例降低提示膜电位下降；0、50、200 μg/mL的rhLF处理口腔癌KB细胞48 h的凋亡率为2.02%、7.60%、48.07%；同时rhLF刺激口腔癌KB细胞能诱导Parp、Caspase-3的激活。结论 rhLF能显著抑制KB细胞的增殖并且诱导其细胞凋亡，其机制与线粒体凋亡途径的激活应激有关。

Recombinant human lactoferrin inhibits proliferation and induces apoptosis in oral cancer KB cells

Objective To investigate the effects of rhLF on the proliferation and apoptosis in oral cancer KB cell line in order to achieve a new effective treatment for oral cancer patients. Methods KB cells were treated with different doses of rhLF (0,12.5,25,50,100,200 μg/mL) for 24,48,72 h and cell viability was detected by CCK-8 assay. Immunofluorescence was used to evaluate the expression of DNA damage associated protein γ-H2AX. Western blotting was performed to examine the expressions of γ-H2AX. Colony formation of the cells was observed after KB cells had been treated with rhLF at 0,200 μg/mL for 14 days. JC-1 assay was used to measure the changes in mitochondrial membrane potential, Annexin V-PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the expressions of Parp and Caspase-3. Results rhLF caused significant suppression of KB cells proliferation. Immunofluorescence showed the increase of γ-H2AX after cells were incubated with rhLF. Western blotting displayed that the expressions of γ-H2AX gradually increased in dose-dependent. Meanwhile, rhLF significantly inhibited KB cells colony formation. Exposure to rhLF (0, 50, 200 μg/mL) after 48 h resulted in 2.02%, 7.60% and 48.07% cell apoptotic rates. Exposure of the cells to increased concentrations of rhLF gradually increased the apoptotic rate and decreased mitochondrial membrane potential. rhLF increased the expression of activated Parp, Caspase-3. Conclusion rhLF can inhibit the proliferation and induce apoptosis of KB cells, the rhLF of which may be associated with the activation of the mitochondrial apoptotic pathway.