干扰素γ和TRAIL联合对HaCaT细胞程序性坏死的影响及机制研究

doi:10.3760/cma.j.issn.0412-4030.2019.05.002

摘要/Abstract

摘要： 【摘要】目的观察干扰素γ（IFN-γ）和肿瘤坏死因子相关凋亡诱导配体（TRAIL）联合对HaCaT人角质形成细胞程序性坏死的诱导作用，并探讨其作用机制。方法 HaCaT细胞分为阴性对照组（不加药物处理）和实验组，实验组分别加入50 μg/L IFN-γ（IFN-γ组）、4 μg/L TRAIL（TRAIL组）、50 μg/L IFN-γ + 4 μg/L TRAIL（细胞因子联合组），或40 μmo/L zVAD预处理1 h后同时加入50 μg/L IFN-γ及4 μg/L TRAIL（zVAD联合组）。作用48 h后，光镜下观察细胞形态，噻唑蓝法检测药物对细胞的增殖抑制作用，双染色法流式细胞仪检测细胞坏死情况，qPCR检测RIP3、MLKL mRNA相对表达量，Western印迹检测细胞RIP1、RIP3、MLKL及其磷酸化蛋白等的表达，免疫荧光染色观察pRIP3、pMLKL在细胞中的表达分布，以2′，7′-二氯荧光素二乙酸酯为荧光探针检测各处理对细胞内活性氧产生的影响。采用SPSS 22进行统计学分析，不同组间观察指标的变化采用单因素方差分析，两两多重比较采用LSD-t检验。结果细胞因子联合组及zVAD联合组作用48 h后HaCaT细胞出现坏死样形态特征。噻唑蓝实验显示作用48 h后，IFN-γ组、TRAIL组、细胞因子联合组、zVAD联合组、阴性对照组细胞存活率分别为73.16% ± 5.71%、81.46% ± 4.68%、72.18% ± 2.93%、69.67% ± 3.24%、100%，5组间差异有统计学意义（F = 24.34，P < 0.001）。细胞因子联合组、zVAD联合组细胞坏死率分别为9.86% ± 1.31%、10.33% ± 2.16%，均高于阴性对照组（5.26% ± 0.91%，t值分别为4.61、5.07，均P < 0.05）。qPCR结果示，细胞因子联合组、zVAD联合组RIP3、MLKL mRNA表达水平明显高于阴性对照组（tRIP3分别为0.99、1.84，tMLKL分别为1.51、2.17，均P < 0.05）。Western印迹结果示，与阴性对照组相比，细胞因子联合组RIP1、RIP3、MLKL及其磷酸化蛋白表达水平升高（均P < 0.05），且zVAD预处理后caspase 8水平下降，上述蛋白表达进一步升高。荧光显微镜示IFN-γ和TRAIL联合作用后，细胞质中可见强绿色点状或团块状荧光斑点（pRIP3），在细胞膜可见明显红色荧光斑点（pMLKL）。活性氧检测示细胞因子联合组平均荧光强度高于阴性对照组，t = 702.00，P < 0.05。结论 IFN-γ和TRAIL可联合诱导HaCaT细胞程序性坏死，并伴有活性氧水平上升。

关键词: 角蛋白细胞；坏死；活性氧；表皮坏死松解症, 中毒性；程序性坏死；受体相互作用蛋白1；受体相互作用蛋白3；混合系激酶区域样蛋白

Abstract: 【Abstract】 Objective To evaluate the inductive effect of interferon-γ （IFN-γ） combined with tumor necrosis factor （TNF）-related apoptosis-inducing ligand （TRAIL） on programmed necrosis of the human immortalized keratinocyte cell line HaCaT, and to explore its mechanisms. Methods In vitro cultured HaCaT cells were divided into several groups: negative control group receiving no treatment, IFN-γ group treated with 50 μg/L IFN-γ, TRAIL group treated with 4 μg/L TRAIL, and cytokine combination group treated with 50 μg/L IFN-γ and 4 μg/L TRAIL or zVAD combination group pretreated with 40 μmo/L zVAD for 1 hour followed by the treatment with 50 μg/L IFN-γ and 4 μg/L TRAIL. After 48-hour treatment, the morphology of HaCaT cells were observed under a light microscope, methyl-thiazolyl-tetrazolium assay was performed to evaluate the inhibitory effect of the treatment on the proliferation of HaCaT cells, and double staining flow cytometry to detect the necrosis of HaCaT cells. Meanwhile, real-time fluorescence-based quantitative PCR （qPCR） was conducted to determine the mRNA expression of receptor interaction protein kinase 3 （RIP3） and mixed lineage kinase domain-like protein （MLKL）, Western blot analysis to determine the expression of RIP1, RIP3, MLKL proteins and their phosphorylated forms （pRIP1, pRIP3, pMLKL）, immunofluorescent staining to observe the distribution of pRIP3 and pMLKL in HaCaT cells, and the level of reactive oxygen species （ROS） in HaCaT cells in the above groups was detected by the fluorescence probe DCFH-DA. Statistical analysis was carried out with SPSS 22 software by using one-way analysis of variance （ANOVA） for comparing indices among different groups, and least significant difference （LSD）-t test for multiple comparisons. Results After 48-hour treatment, HaCaT cells in the cytokine combination group and zVAD combination group showed necrosis-like morphologic features. Methyl-thiazolyl-tetrazoliumassay revealed significant differences in the survival rate of HaCaT cells among the IFN-γ group, TRAIL group, cytokine combination group, zVAD combination group and negative control group （73.16% ± 5.71%, 81.46% ± 4.68%, 72.18% ± 2.93%, 69.67% ± 3.24% and 100%, respectively; F = 24.34, P < 0.001）. The necrosis rate of HaCaT cells was notably higher in the cytokine combination group and zVAD combination group （9.86% ± 1.31%, 10.33% ± 2.16%, respectively） than in the negative control group （5.26% ± 0.91%, t = 4.61, 5.07, respectively, both P < 0.05）. qPCR revealed that the mRNA expression of RIP3 and MLKL significantly increased in the cytokine combination group and zVAD combination group compared with the negative control group （tRIP3 = 0.99, 1.84, tMLKL = 1.51, 2.17, respectively, all P < 0.05）. Western blot analysis suggested that the protein expression of RIP1, RIP3, MLKL, pRIP1, pRIP3 and pMLKL significantly increased in the cytokine combination group compared with the negative control group （all P < 0.05）, and the zVAD combination group showed significantly decreased caspase 8 expression and increased expression of the above proteins compared with the cytokine combination group. Fluorescence microscopy showed that enhanced green dot-like or clump-like fluorescent spots （representing pRIP3） could be observed in the cytoplasm, and red fluorescent spots （representing pMLKL） could be seen on the cell membrane in the cytokine combination group. The average fluorescence intensity of ROS was significantly higher in the cytokine combination group than in the negative control group （t = 702.00, P < 0.05）. Conclusion IFN-γ combined with TRAIL can induce the programmed necrosis of HaCaT cells with increased level of ROS.

Key words: Keratinocytes, Necrosis, Reactive oxygen species, Epidermal necrolysis, toxic, Programmed necrosis, Receptor interaction protein kinase 1, Receptor interaction protein kinase 3, Mixed lineage kinase domain?like protein

中图分类号:

R758.25

寿艳红张臻骆肖群陈圣安李峰朱小华林尽染秦海红杜鹃陈荪奕杨永生徐金华. 干扰素γ和TRAIL联合对HaCaT细胞程序性坏死的影响及机制研究[J]. 中华皮肤科杂志, 2019, 52(5): 302-309.

Shou Yanhong, Zhang Zhen, Luo Xiaoqun, Chen Sheng′an, Li Feng, Zhu Xiaohua, Lin Jinran, Qin Haihong, Du Juan, Chen Sunyi, Yang Yongsheng, Xu Jinhua . Effect of interferon-γ combined with tumor necrosis factor-related apoptosis-inducing ligand on programmed necrosis of HaCaT cells and its mechanisms[J]. Chinese Journal of Dermatology, 2019, 52(5): 302-309.

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