从TOCP暴露鸡脊髓组织中筛选OPIDN诱发相关蛋白

发布时间：2018-07-03 13:52

本文选题：三磷甲苯磷酸酯 + 苯甲基磺酰氟　；参考：《大连医科大学》2012年硕士论文

【摘要】：目的：利用PMSF前干预对OPIDN影响特点，通过双向电泳和质谱分析技术，构建OPIDN相关蛋白信息谱，筛选并鉴定TOCP诱发OPIDN相关差异表达蛋白，为探讨TOCP诱发OPIDN作用机制提供靶蛋白依据。方法：取罗曼鹤鸡56只，随机分为低剂量TOCP（750mg/kg）染毒组、高剂量TOCP（1000mg/kg）染毒组、给予PMSF（40mg/kg）后再投TOCP（1000mg/kg）的PMSF前干预组以及对照组，每组14只，分别观察四组鸡迟发型神经毒性行为症状，并于染毒后第5天和20天，从各组8只鸡中取脊髓组织用于双向电泳和质谱分析，而每组剩余6只鸡继续观察迟发性神经毒性症状，第23天观察结束；OPIDN症状观察结束后，处死实验动物，选取高剂量组鸡脊髓组织进行毒理形态观察。双向电泳原始文件用imagemaster2Dplatinum软件搜索相应的数据库，蛋白表达差异2倍以上或0.5倍以下视为差异显著点。利用质谱分析技术对差异表达蛋白点鉴定，通过计算机MASCOT软件的MS MS离子方式对肽质量指纹图谱和分子量、等电点数据进行分析，于蛋白质数据库中搜索与之匹配的相关蛋白，同时查询其功能。肽质量指纹图谱容许误差0.3Da，分子量容许误差±20%。结果：对神经症状观察的结果显示，两组TOCP暴露鸡从染毒后第5天开始出现轻度迟发性神经毒性症状，并随时间推移其神经毒性症状逐渐加重，第20天这些鸡的平均迟发性神经毒性症状评分值基本达到高峰。尤其，高剂量组鸡的迟发性神经毒性症状较为严重。在染毒23天，高剂量TOCP组的6只中全部出现了OPIDN症状，而低剂量TOCP组的6只中只有3只出现了OPIDN症状。相反，PMSF前干预组和对照组鸡没有出现迟发性神经毒性症状。通过组织病理学对TOCP暴露23天的鸡神经组织观察，可见神经元突起消失，髓鞘肿胀和脱髓鞘，神经元坏死，胶原纤维增生等，出现具有特征性的病理学变化。进一步从病理学角度证实了TOCP诱发鸡OPIDN模型的可靠性。利用双向电泳技术筛选OPIDN相关差异表达蛋白点信息，从TOCP暴露鸡脊髓神经组织中共筛选136个与OPIDN相关差异表达蛋白点，其中上调点有23个，下调点有113个。利用质谱分析技术，，从鸡脊髓组织相关差异表达蛋白点中鉴定了14个可能与OPIDN相关的靶标蛋白，其中下调蛋白13个： cofilin-1-B、Stathmin蛋白、髓鞘碱性蛋白(MBP)、突触核蛋白、热休克蛋白beta-1、胰蛋白激酶A、磷酸酪氨酸蛋白磷酸酶、苹果酸脱氢酶、精氨酸激酶、一型细胞骨架蛋白、Vesicle amine transport protein1、塔尔羊蛋白5、电子传递黄素蛋白；上调调蛋白1个：神经丝蛋白。结论：1000mg/kg TOCP暴露和40mg/kg PMSF前干预可获得稳定的鸡OPIDN模型和PMSF干预模型。通过双向电泳和质谱分析技术从鸡脊髓组织中筛选和鉴定了14个可能与OPIDN密切相关蛋白。其中下调蛋白包括5个神经组织相关蛋白，4个蛋白酶类蛋白和4个其他相关蛋白。上调蛋白1个，为神经丝蛋白。
[Abstract]:Objective: to construct the OPIDN-associated protein information spectrum by using the characteristics of PMSF pre-intervention on OPIDN, and to screen and identify the differentially expressed OPIDN related proteins induced by TOCP. In order to investigate the mechanism of TOCP induced OPIDN, the target protein was provided. Methods: Fifty-six Romanhe chickens were randomly divided into low dose (750mg/kg) group, high dose TOCP (1000mg/kg) group, PMSF (40mg/kg) treated group and control group. 14 chickens in each group were observed the symptoms of delayed neurotoxic behavior in each group. On the 5th and 20th day after exposure, spinal cord tissues were taken from 8 chickens in each group for two-dimensional electrophoresis and mass spectrometry analysis. The remaining 6 chickens in each group continued to observe the symptoms of delayed neurotoxicity, and on the 23rd day, the OPIDN symptoms were observed after the end of the observation. The experimental animals were killed and the high dose group was selected to observe the morphology of spinal cord. The original file was searched by imagemaster2Dplatinum software. The difference of protein expression was more than 2 times or less than 0.5 times. The differentially expressed protein spots were identified by mass spectrometry. The peptide mass fingerprints, molecular weight and isoelectric point data were analyzed by MS ion mode of computer MASCOT software, and the corresponding proteins were searched in the protein database. At the same time query its function. The allowable error of peptide fingerprint was 0.3 Daa, and that of molecular weight 卤20. Results: the neurotoxic symptoms of the two groups of TOCP exposed chickens began to appear mild delayed neurotoxic symptoms from the 5th day after exposure, and the neurotoxic symptoms increased gradually over time. On day 20, the average delayed neurotoxicity symptom score of these chickens reached the peak. Especially, the symptoms of delayed neurotoxicity in high dose group were more serious. After 23 days of exposure, OPIDN symptoms were found in all of the 6 rats in the high dose TOCP group and only 3 in the low dose TOCP group. On the contrary, there were no delayed neurotoxic symptoms in the intervention group and control group before PMSF. Through histopathological observation of chicken nerve tissue exposed to TOCP for 23 days, the neuronal process disappeared, myelin swelling and demyelination, neuronal necrosis, collagen fiber proliferation, and so on, were observed. The reliability of OPIDN model induced by TOCP was confirmed by pathology. A total of 136 differentially expressed protein sites associated with OPIDN were screened from nerve tissue of chicken spinal cord exposed to TOCP with 23 up-regulation points and 113 down-regulation points. Using mass spectrometry, 14 target proteins related to OPIDN were identified from differentially expressed protein sites in chicken spinal cord tissue. Among them, 13 down-regulated proteins were cofilin-1-BnStathmin protein, myelin basic protein (MBP) and synaptophysin. Heat shock protein beta-1, trypsin kinase A, phosphotyrosine protein phosphatase, malate dehydrogenase, arginine kinase, type I cytoskeleton protein amine transport protein 1, Tal sheep protein 5, electron transfer protein 5, upper modulator protein 1: neurofilament protein. Conclusion the stable chicken OPIDN model and PMSF intervention model can be obtained after 1: 1000 mg / kg TOCP exposure and 40mg/kg PMSF intervention. Fourteen proteins that may be closely related to OPIDN were screened and identified from chicken spinal cord by two-dimensional electrophoresis and mass spectrometry. The down-regulated proteins include 5 nerve tissue related proteins, 4 protease-like proteins and 4 other related proteins. Upregulation protein was a neurofilament protein.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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