三邻甲苯磷酸酯暴露鸡脊髓组织中Cofilin-1b的差异表达

The differential expression of cofilin-1b in the spinal cord tissue of TOCP-treated hens

目的从三邻甲苯磷酸酯(TOCP)暴露鸡的脊髓组织中筛选可能与调控微丝解聚作用相关的差异表达蛋白,为探讨有机磷化合物诱发的迟发性神经毒性(OPIDN)作用机制提供靶蛋白依据。方法 42只罗曼鹤母鸡随机分成1000 mg/kg TOCP组、预先给予40 mg/kg苯甲基磺酰氟(PMSF)后再投1000 mg/kg TOCP的干预组和生理盐水对照组,每组14只。染毒第5和20天,每组分别处死4只鸡,低温环境下分离脊髓,提取总蛋白。利用双向电泳结合质谱分析技术,筛选和鉴定可能与调控微丝解聚作用相关的差异表达蛋白。结果 TOCP组鸡于染毒第7日前后出现进行性共济失调和肌无力等OPIDN典型症状,起病从下肢远端部分开始且病变程度随时间逐渐加重直至全瘫,而其他组鸡在实验观察期间未见OPIDN症状。TOCP组鸡于暴露第5天,分别与对照组和PMSF前干预组比较,其脊髓组织肌动蛋白解聚因子Cofilin-1b分别下调3.4倍和2.8倍,且有统计学意义(差异表达<0.5或差异表达>2),而PMSF前干预组与对照组比较,鸡脊髓组织Cofilin-1b的表达差异无统计学意义。在TOCP暴露第20天,TOCP组鸡脊髓组织Cofilin-1b表达与其他两组比较尽管有下降趋势,但无显著性变化。结论 TOCP暴露能导致鸡脊髓神经组织Cofilin-1b表达在早期显著下调,且该蛋白表达下调可能与微丝骨架结构紊乱及其OPIDN诱发机制有关。

Objective To screen differently expressed proteins related to regulate depolymerization of microfilament in the spinal cord of hens exposed to tri-orhto-cresyl phosphate（TOCP） so as to provide target protein evidence for exploring the mechanisms of organophosphorus compounds（OPs） inducing delayed neurtoxicity（OPIDN）.Methods Forty two Luoman He hens were randomly divided into three groups and 14 hens in each group,TOCP group was treated with 1000 mg/kg TOCP.Hens in PMSF interfering group were given with TOCP after 40 mg/kg phenylmethysulfonyl fluoride（PMSF） administration.The control group was received with normal saline.Hens were sacrificed on the 5th and 20th day.Spinal cord was decoherenced and homogenated at low temperature,and the total proteins were Abstracted.The differently expressed proteins related to regulate depolymerization of microfilament were screen by two-dimensional electrophoresis and mass spectroscopy（MS）.Results Hens in TOCP group showed OPIDN typical signs（progressive ataxia and hypotonia） about 7 days after TOCP exposure,the sydrom began to appear from the distal part of lower extremity and the damage degree aggravated gradully to pamplegia,while OPIDN symtoms in the other two groups were not observed in the experimental period.The result of early stage（the 5th day） show that the actin depolymerizing factor Cofilin-1b expression was down-regulated 3.4 times and 2.8 times respectively in TOCP group compared with the control and PMSF intervention group,and had significant difference.However,the difference between control group and PMSF intervention group was not significant.On the 20th day,the expression of Cofilin-1b was down-regulated between TOCP group and the other two groups,but the difference was not significant.Conclusion TOCP downregulate significantly the Coflilin-1b expression in the tissue of spinal cord.It indicates that the down-regulated Coflilin-1b expression may be related to excess poylymerization of microfilament and causative mechanism of OPIDN.