CAJ | 학술논문

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.
목적 탐토취-ADP-핵당기화재Cr(Ⅵ)치세포손해중적작용.방법 선용전기건립적취-ADP-핵당수해매(PARG)결함적인지기관상피세포(16HBE세포)작위연구대상,선용불동제량적Cr(Ⅵ)분별염독,병처리정상16HBE세포화PARG결함세포24 h,동시설립용제대조조,비교량충세포대Cr(Ⅵ)독작용적차이;재차기출상,선취5.0 μmol/L적Cr(Ⅵ)작위염독제량,염독처리량충세포후,제취세포총단백진행쌍향형광차이응효전영(2 D-DIGE)분석,계산비교량충세포염독전、후적총단백표체차이,대차이단백점진행기질보조격광해흡전리비행시간천련질보(MALDI-TOF-MS/MS)감정,병진일보사용Western blot험증.결과 Cr(Ⅵ)작용후,PARG결함세포적존활교정상16HBE세포다,당작용제량체도5.0 μmoL/L시,16HBE세포화PARG결함세포적존활솔분별위(59.67±6.43)％화(82.00±6.25)％,량자지간적차이유통계학의의(t=-4.32,P＜0.05);2D-DIGE분석비교정상16HBE세포여PARG결함세포Cr(Ⅵ)염독전、후단백차이,공사선차성공감정출18개단백,저사단백적공능섭급유지세포형태、삼여능량대사、DNA손상수복이급기인표체조공.Western blot성공험증출차이표체단백cofilin-1,기재염독후적16HBE세포중표체상조,상대표체량위1.41 ±0.04,대조조표체량위1.00±0.01,차이유통계학의의(t=-18.00,P＜0.05).재PARG결함세포표체차이무통계학의의(t=-8.61,P＞0.05).결론 감정출적차이단백대다여종류발생밀절상관,추측취-ADP-핵당기화반응가통과억제Cr(Ⅵ)유도적종류발생종이대항Cr(Ⅵ)적세포독성,저위천명취-ADP-핵당기화재Cr(Ⅵ)치세포손해중적작용궤제제공료중요적삼고의거.
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 cells had no statistically significant difference(t =-8.61,P ＞ 0.05).Conclusion Most of the identified differential proteins are closely related to tumorigenesis,suggesting that poly-ADP-ribosylation reaction may resist the cytotoxicity of Cr(Ⅵ) by inhibiting Cr (Ⅵ) induced tumorigenesis,which provides important reference data to clarify the mechanisms of poly-ADPribosylation in Cr (Ⅵ) induced cell damage.