OPIDN致鸡大脑脑型肌酸激酶差异表达

Differential expression of CK-BB in brain tissue of hens with OPIDN

目的探讨有机磷化合物致迟发性神经毒性(OPIDN)鸡脑组织中脑型肌酸激酶同工酶(CK-BB)表达变化。方法罗曼鹤母鸡24只,随机分成三磷甲苯磷酸酯(TOCP)染毒组(1 000 mg/kg)、苯甲基磺酰氟(PMSF)干预组(在染毒之前给予40 mg/kg PMSF)和对照组(生理盐水),每组8只。染毒第5和第21天,每组分别处死8只动物,分离大脑,提取总蛋白;用双向电泳和质谱分析技术,筛选相关差异表达蛋白。结果蛋白双向电泳结果显示,在染毒第5天,TOCP组与对照组、PMSF干预组与对照组的CK-BB蛋白点灰度比值分别为0.858 3和0.913 3,各组间差异无统计学意义(P>0.05);染毒第21天,TOCP组和PMSF干预组与对照组差异蛋白点CK-BB灰度比值分别为0.327 5与0.675 7,前者表达下调达3.0倍;对该蛋白点的质谱分析结果在Swiss-por数据库中进行比对,其与鸡种属、蛋白序列号为gi|45384340的creatine kinase B-type(CK-BB)蛋白的肽段匹配数为26,序列覆盖率为75%,评分值为1 603(可信度高),显示该蛋白点即为CK-BB蛋白。结论 OPIDN可致鸡大脑组织CK-BB蛋白表达显著下调,其变化可能与OPIDN模型动物神经组织损伤有关。

Objective To detect differential expressions of brain type isoenzyme of creatine kinase （ CK-BB ） in the brain of hens with organophophors compounds induced delayed nerutoxicity （OPIDN）and to provide target protein evidence for diagnosis of POIDN. Methods Twenty-four Roman hens were randomly divided into three groups ： treated group（ 1 000 mg/kg triorthocresyl phosphate, TOCP）, intervened group （40 mg/kg phenylmethanesul fonyl fluoride [ PMSF] before TOCP treatment）, and control group （tap water）. Eight hens in each group were sacrificed at day 5 and day 20 after the treatments;then, the brain tissue of the hens were seperated at low temperature and total protein of the brain tissue were extracted. With solid phase PH gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and two-dimensional electrophoresis and mass spectrometry, we detected differently expressed CK-BB protein. Results Compared with the control group, the difference in CK-BB expression was not significant at day 5 （P 〈 0. 05 ）,while at day 20, the gray level ratio of CK-BB expression of treated-group and intervened-group were 0. 327 5 and 0. 675 7 ,with a three-fold down-regulation in the treated group. Mass spectrometry and Swiss-por data anal- ysis identified CK-BB spot with peptide segments of 26, sequence coverage of 75%, and the assessment value of 1 603 （ ≥45 for high reliability）in relation to the species of the hen and protein sequence number gil45384340 of CK-BB. Conclusion The expression of CK-BB is down-regulated in the brain of hens with OPIDN, which may related to brain injury induced by OPIDN.