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中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (06) : 346 -353. doi: 10.3877/cma.j.issn.1674-1366.2017.06.005

所属专题: 文献

基础研究

重组人乳铁蛋白诱导人口腔癌Tca8113细胞自噬及凋亡
肖莉1,(), 张继斌1, 李宝霞2   
  1. 1. 511400 广州市番禺区中心医院
    2. 510600 广州,中山大学肿瘤防治中心,华南肿瘤国家重点实验室
  • 收稿日期:2017-04-06 出版日期:2017-12-01
  • 通信作者: 肖莉
  • 基金资助:
    广东省科技计划(2013B021800049)

Recombinant human lactoferrin induces autophagy and apoptosis in human oral cancer Tca8113 cells

Li Xiao1,(), Jibin Zhang1, Baoxia Li2   

  1. 1. Panyu Central Hospital, Guangzhou 511400, China
    2. Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou 510060, China
  • Received:2017-04-06 Published:2017-12-01
  • Corresponding author: Li Xiao
  • About author:
    Corresponding author: Xiao Li, Email:
引用本文:

肖莉, 张继斌, 李宝霞. 重组人乳铁蛋白诱导人口腔癌Tca8113细胞自噬及凋亡[J]. 中华口腔医学研究杂志(电子版), 2017, 11(06): 346-353.

Li Xiao, Jibin Zhang, Baoxia Li. Recombinant human lactoferrin induces autophagy and apoptosis in human oral cancer Tca8113 cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(06): 346-353.

目的

研究重组人乳铁蛋白(rhLF)对人口腔癌Tca8113细胞增殖及凋亡的影响。

方法

MTT法检测不同浓度的rhLF作用细胞不同时间对其增殖的影响,并分别用不同浓度的rhLF处理细胞后观察其对集落克隆形成的影响;不同浓度的rhLF处理细胞后线粒体膜电位的变化。采用免疫荧光技术检测rhLF处理前后自噬体的形成情况。免疫蛋白印迹法(Western blot)检测不同浓度rhLF处理细胞后细胞自噬相关蛋白P62和LC3表达变化,Annexin Ⅴ-PI双染色检测rhLF对细胞凋亡的影响;Western blot检测不同浓度的rhLF处理细胞后聚腺苷二磷酸-核糖聚合酶(PARP)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达变化。所有数据采用SPSS 13.0统计软件包进行统计学分析。

结果

rhLF对口腔癌Tca8113细胞的增殖及存活有明显的增殖抑制作用,并呈药物浓度和时间依赖性,且各药物处理组与对照组相比有统计学意义(P<0.05)。rhLF抑制口腔癌Tca8113细胞克隆形成;JC-1荧光检测显示口腔癌Tca8113细胞经rhLF处理后其线粒体膜电位下降;rhLF处理后形成自噬体,不同浓度rhLF处理细胞后细胞自噬相关蛋白P62表达下降及LC3-Ⅱ表达增加,0、100、200 μg/ml的rhLF处理口腔癌Tca8113细胞48 h的凋亡率为3.9%、8.2%、42.6%,组间差异有统计学意义(P<0.05)。Western blot结果显示,rhLF处理口腔癌Tca8113细胞后PARP、Caspase-3被激活。

结论

rhLF能显著抑制Tca8113细胞的增殖并诱导其细胞凋亡,其机制与线粒体凋亡途径的激活应激有关。

Objective

To investigate the effect of recombinate human lactoferrin (rhLF) on the proliferation and apoptosis of human oral cancer cell line Tca8113 in vitro.

Methods

Cells were exposed to different doses of rhLF at different times, and cell viability was evaluated by MTT. Colony formation was observed and mitochondrial membrane potential was measured by JC-1 assay. Immunofluorescence was used to detect the formation of autophagic vacuoles Western blot was performed to examine the expression of P62, LC3 and the protein expression of PARP and Caspase-3. Annexin Ⅴ-PI staining was used to assess the effect of rhLF on apoptosis. All data were analyzed by SPSS 13.0 software package.

Results

The proliferation and survival of Tca8113 cells were significantly inhibited by rhLF in a dose- and time - dependent manner, and the difference between the test groups and the control group was statistically significant (P<0.05) . Specifically, cell colony formation of Tca8113 was inhibited by rhLF. Mitochondrial membrane potential was gradually decreased with exposure to rhLF. The autophagosome was observed and the expression of autophagy-related protein P62 and LC3-Ⅱ was increased after rhLF treatment. The apoptosis rate of Tca8113 cells treated with rhLF at 0, 100, 200 μg/ml for 48 h was 3.9%, 8.2% and 42.6%, respectively. There was significant difference between groups (P<0.05) . Western blot results showed that PARP and Caspase-3 were activated after Tca8113 cells were treated with rhLF.

Conclusions

RhLF was found to significantly inhibit the proliferation of Tca8113 cells and induce apoptosis of Tca8113 cells, and its mechanism was related to the activation of the mitochondrial apoptotic pathway.

表1 不同浓度rhLF处理后对Tca8113细胞的增殖抑制情况(%,±s
图1 Hoechst 33258染色检测不同浓度rhLF诱导Tca8113细胞48 h的细胞凋亡情况(荧光显微镜 低倍放大)
图2 不同浓度重组人乳铁蛋白抑制Tca8113细胞14 d后集落克隆的形成情况
图3 不同浓度rhLF处理48 h对口腔癌Tca8113细胞线粒体膜电位的影响(JC-1染色)
图4 rhLF诱导Tca8113细胞发生自噬
图5 rhLF诱导Tca8113细胞凋亡
图6 不同浓度rhLF处理Tca8113细胞48 h后激活Caspases-3和PARP
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