CAJ | 학술논문

目的从三邻甲苯磷酸酯(TOCP)染毒鸡大脑组织中筛选迟发性神经毒性(OPIDN)相关差异表达蛋白,为进一步探讨OPIDN机制提供靶标蛋白.方法成年罗曼鹤母鸡32只随机分成1000mg/kg TOCP组(染毒组)、先给40mg/kg PMSF后24 h再给予1000 mg/kg TOCP组(PMSF干预组)、给予40 mg/kg PMSF组(PMSF组)和给予生理盐水的对照组,每组8只.一次性染毒后第5天处死动物,低温环境下分离大脑,匀浆,高速离心提取总蛋白.通过双向凝胶电泳获得完整的全蛋白质图谱,运用图像分析软件(Image Master 2D)对银离子染色的电泳图谱进行分析,并对所选取的蛋白点进行质谱鉴定.结果染毒组、PMSF干预组和PMSF组鸡大脑组织总蛋白中,分别检测到1185、1294和1063个蛋白点,对照组检测到1332个蛋白点.与对照组比较,染毒组、PMSF干预组和PMSF组匹配率分别为78.32%、79.56%、80.93%.与对照组比较,染毒组差异表达蛋白点有235个,其中,上调点有158个,下调点有77个.根据PMSF的作用特点,在TOCP组中选择与对照组比较差异明显且与PMSF干预组无明显差异的蛋白匹配点,共获得有102个.其中差异表达4倍以上且与PMSF组二次匹配差异无统计学意义的蛋白点有13个.对这些蛋白点做进一步质谱分析和鉴定,获得7个蛋白:homer-1b、Destrin蛋白、热休克蛋白70、真核翻译起始因子、蛋白酶体α1亚基、乳酸脱氢酶B、代谢性谷氨酰胺合成酶.结论 TOCP染毒鸡大脑组织中有112个差异表达蛋白点,可能与OPIDN诱发有关,其中13个差异表达蛋白点可能与OPIDN诱发有密切的关联性.质谱鉴定出7个可能与OPIDN机制关联的蛋白.
목적 종삼린갑분린산지(TOCP)염독계대뇌조직중사선지발성신경독성(OPIDN)상관차이표체단백,위진일보탐토OPIDN궤제제공파표단백.방법 성년라만학모계32지수궤분성1000mg/kg TOCP조(염독조)、선급40mg/kg PMSF후24 h재급여1000 mg/kg TOCP조(PMSF간예조)、급여40 mg/kg PMSF조(PMSF조)화급여생리염수적대조조,매조8지.일차성염독후제5천처사동물,저온배경하분리대뇌,균장,고속리심제취총단백.통과쌍향응효전영획득완정적전단백질도보,운용도상분석연건(Image Master 2D)대은리자염색적전영도보진행분석,병대소선취적단백점진행질보감정.결과 염독조、PMSF간예조화PMSF조계대뇌조직총단백중,분별검측도1185、1294화1063개단백점,대조조검측도1332개단백점.여대조조비교,염독조、PMSF간예조화PMSF조필배솔분별위78.32%、79.56%、80.93%.여대조조비교,염독조차이표체단백점유235개,기중,상조점유158개,하조점유77개.근거PMSF적작용특점,재TOCP조중선택여대조조비교차이명현차여PMSF간예조무명현차이적단백필배점,공획득유102개.기중차이표체4배이상차여PMSF조이차필배차이무통계학의의적단백점유13개.대저사단백점주진일보질보분석화감정,획득7개단백:homer-1b、Destrin단백、열휴극단백70、진핵번역기시인자、단백매체α1아기、유산탈경매B、대사성곡안선알합성매.결론 TOCP염독계대뇌조직중유112개차이표체단백점,가능여OPIDN유발유관,기중13개차이표체단백점가능여OPIDN유발유밀절적관련성.질보감정출7개가능여OPIDN궤제관련적단백.
Objective To screen the proteins with differential expression levels in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate (TOCP),and to provide target proteins for studying the mechanism of organophosphoms ester-induced delayed neurotoxicity (OPIDN). Methods Thirty two adult Roman hens were randomly divided into four groups: TOCP group was exposed to 1000 mg/kg TOCP, PMSF group was exposed to 40 mg/kg PMSF, PMSF plus TOCP group was exposed to 40 mg/kg PMSF and after 24 h exposed to 1000 mg/kg TOCP, control group was exposed to normal saline. All hens exposed to chemicals by gastro-intestine for 5 days were sacrificed, and the cerebral tissue were dissected and homogenized in ice bath. Total proteins extracted from the cerebral tissue were separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The 2-DE maps were visualized after silver staining and analyzed by Image Master 2D software. At last ,the expressed protein spots were identified by Mass spectrometry. Results From total proteins in TOCP group, the PMSF plus TOCP group and PMSF group, 1185, 1294 and 1063 spots were detected, respectively. One thousand three hundred thirty two spots from total proteins in control group were detected. The match rates of protein spots in TOCP group, the PMSF plus TOCP group and PMSF group were 78.32 %, 79.56 % and 80.93%, respectively. There were 235 protein spots with differential expression levels between TOCP group and control group, which included 158 up regulation spots and 77 down regulation spots. According to the PMSF features, there were 102 spots with differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF plus TOCP group, among them there were 13 spots with 4 fold differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF group. Seven protein spots (homer-1b, Destrin, heat shock protein 70, eukaryotic translation initiation factors, proteasome α1 subunit, lactate dehydrogenase B, glutamine synthetase) were detected by Mass spectrometry. Conclusion There are 112 protein spots with differential expression levels of the cerebral tissue in TOCP group,which may be related to OPIDN,among them 13 protein spots with differential expression levels are associated closely with OPIDN.Seven protein spots detected by Mass spectrometry may be related to the mechanism induced by OPIDN.