摘要
目的 探讨聚-ADP-核糖基化在Cr（Ⅵ）致细胞损害中的作用.方法 选用前期建立的聚-ADP-核糖水解酶（PARG）缺陷的人支气管上皮细胞（16HBE细胞）作为研究对象,选用不同剂量的Cr（Ⅵ）分别染毒,并处理正常16HBE细胞和PARG缺陷细胞24 h,同时设立溶剂对照组,比较两种细胞对Cr（Ⅵ）毒作用的差异;在此基础上,选取5.0 μmol/L的Cr（Ⅵ）作为染毒剂量,染毒处理两种细胞后,提取细胞总蛋白进行双向荧光差异凝胶电泳（2 D-DIGE）分析,计算比较两种细胞染毒前、后的总蛋白表达差异,对差异蛋白点进行基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-MS/MS）鉴定,并进一步使用Western blot验证.结果 Cr（Ⅵ）作用后,PARG缺陷细胞的存活较正常16HBE细胞多,当作用剂量达到5.0 μmoL/L时,16HBE细胞和PARG缺陷细胞的存活率分别为（59.67±6.43）％和（82.00±6.25）％,两者之间的差异有统计学意义（t=-4.32,P＜0.05）;2D-DIGE分析比较正常16HBE细胞与PARG缺陷细胞Cr（Ⅵ）染毒前、后蛋白差异,共筛选且成功鉴定出18个蛋白,这些蛋白的功能涉及维持细胞形态、参与能量代谢、DNA损伤修复以及基因表达调控.Western blot成功验证出差异表达蛋白cofilin-1,其在染毒后的16HBE细胞中表达上调,相对表达量为1.41 ±0.04,对照组表达量为1.00±0.01,差异有统计学意义（t=-18.00,P＜0.05）.在PARG缺陷细胞表达差异无统计学意义（t=-8.61,P＞0.05）.结论 鉴定出的差异蛋白大多与肿瘤发生密切相关,推测聚-ADP-核糖基化反应可通过抑制Cr（Ⅵ）诱导的肿瘤发生从而对抗Cr（Ⅵ）的细胞毒性,这为阐明聚-ADP-核糖基化在Cr（Ⅵ）致细胞损害中的作用机制提供了重要的参考依据.
Objective To investigate the effect of poly-ADP-ribosylation in hexavalent chromium Cr （Ⅵ） induced cell damage.Methods The study object,poly （ADP-ribose） glycohydrolase （PARG） deficient human bronchial epithelial cells （16HBE cells）,was constructed previously by our research group.Normal 16HBE cells and PARG-deficient cells were treated with different doses of Cr （Ⅵ） for 24 h to compare the differences to Cr （Ⅵ） toxicity,meanwhile set up the solvent control group.On this basis,5.0 μmol/L of Cr （Ⅵ） was selected as the exposure dose,after the exposure treatment,total proteins of both cells were extracted for two dimension fluorescence difference gel electrophoresis （2D-DIGE） separation,statistically significant differential protein spots were screened and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS/MS）,and further validated by Western blot.Results After Cr （Ⅵ） treatment,the survival rate of PARG-deficient cells was higher than normal 16HBE cells.When the doses reached up to 5.0 μmol/L,the survival rate of 16HBE cells and PARG-deficient cells were respectively （59.67 ± 6.43） % and （82.00 ± 6.25） %,the difference between which was significant （t =-4.32,P 〈 0.05）.18 protein spots were selected and successfully identified after 2D-DIGE comparison of differential proteins between normal 16HBE cells and PARG-deficient cells before and after exposure.The function of those proteins was involved in the maintenance of cell shape,energy metabolism,DNA damage repair and regulation of gene expression.The differential expression of cofilin-1 was successfully validated by Western blot.The expression level of cofilin-1 in the 16HBE cells increased after Cr （Ⅵ） exposure with the relative expression quantity of 1.41 ± 0.04 in treated group and 1.00 ±0.01 in control group,the difference of which was statistically significant （t =-18.00,P 〈 0.05）,while the expression level in PARG-deficient cel
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2014年第8期720-725,共6页
Chinese Journal of Preventive Medicine
基金
国家自然科学基金（81001261,81370080）
深圳市重点实验室项目（CXB201104220031A）
深圳市科技研发资金（JCYJ20130329103949642）
