本文選題：三磷甲苯磷酸酯 + 苯甲基磺酰氟��； 參考：《大連醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：利用PMSF前干預(yù)對(duì)OPIDN影響特點(diǎn)，通過(guò)雙向電泳和質(zhì)譜分析技術(shù)，構(gòu)建OPIDN相關(guān)蛋白信息譜，篩選并鑒定TOCP誘發(fā)OPIDN相關(guān)差異表達(dá)蛋白，為探討TOCP誘發(fā)OPIDN作用機(jī)制提供靶蛋白依據(jù)。 方法：取羅曼鶴雞56只，隨機(jī)分為低劑量TOCP（750mg/kg）染毒組、高劑量TOCP（1000mg/kg）染毒組、給予PMSF（40mg/kg）后再投TOCP（1000mg/kg）的PMSF前干預(yù)組以及對(duì)照組，每組14只，分別觀察四組雞遲發(fā)型神經(jīng)毒性行為癥狀，并于染毒后第5天和20天，從各組8只雞中取脊髓組織用于雙向電泳和質(zhì)譜分析，而每組剩余6只雞繼續(xù)觀察遲發(fā)性神經(jīng)毒性癥狀，第23天觀察結(jié)束；OPIDN癥狀觀察結(jié)束后，處死實(shí)驗(yàn)動(dòng)物，選取高劑量組雞脊髓組織進(jìn)行毒理形態(tài)觀察。 雙向電泳原始文件用imagemaster2Dplatinum軟件搜索相應(yīng)的數(shù)據(jù)庫(kù)，蛋白表達(dá)差異2倍以上或0.5倍以下視為差異顯著點(diǎn)。利用質(zhì)譜分析技術(shù)對(duì)差異表達(dá)蛋白點(diǎn)鑒定，通過(guò)計(jì)算機(jī)MASCOT軟件的MS MS離子方式對(duì)肽質(zhì)量指紋圖譜和分子量、等電點(diǎn)數(shù)據(jù)進(jìn)行分析，于蛋白質(zhì)數(shù)據(jù)庫(kù)中搜索與之匹配的相關(guān)蛋白，同時(shí)查詢其功能。肽質(zhì)量指紋圖譜容許誤差0.3Da，分子量容許誤差±20%。 結(jié)果：對(duì)神經(jīng)癥狀觀察的結(jié)果顯示，兩組TOCP暴露雞從染毒后第5天開(kāi)始出現(xiàn)輕度遲發(fā)性神經(jīng)毒性癥狀，并隨時(shí)間推移其神經(jīng)毒性癥狀逐漸加重，第20天這些雞的平均遲發(fā)性神經(jīng)毒性癥狀評(píng)分值基本達(dá)到高峰。尤其，高劑量組雞的遲發(fā)性神經(jīng)毒性癥狀較為嚴(yán)重。在染毒23天，高劑量TOCP組的6只中全部出現(xiàn)了OPIDN癥狀，而低劑量TOCP組的6只中只有3只出現(xiàn)了OPIDN癥狀。相反，PMSF前干預(yù)組和對(duì)照組雞沒(méi)有出現(xiàn)遲發(fā)性神經(jīng)毒性癥狀。通過(guò)組織病理學(xué)對(duì)TOCP暴露23天的雞神經(jīng)組織觀察，可見(jiàn)神經(jīng)元突起消失，髓鞘腫脹和脫髓鞘，神經(jīng)元壞死，膠原纖維增生等，出現(xiàn)具有特征性的病理學(xué)變化。進(jìn)一步從病理學(xué)角度證實(shí)了TOCP誘發(fā)雞OPIDN模型的可靠性。利用雙向電泳技術(shù)篩選OPIDN相關(guān)差異表達(dá)蛋白點(diǎn)信息，從TOCP暴露雞脊髓神經(jīng)組織中共篩選136個(gè)與OPIDN相關(guān)差異表達(dá)蛋白點(diǎn)，其中上調(diào)點(diǎn)有23個(gè)，下調(diào)點(diǎn)有113個(gè)。利用質(zhì)譜分析技術(shù)，，從雞脊髓組織相關(guān)差異表達(dá)蛋白點(diǎn)中鑒定了14個(gè)可能與OPIDN相關(guān)的靶標(biāo)蛋白，其中下調(diào)蛋白13個(gè)： cofilin-1-B、Stathmin蛋白、髓鞘堿性蛋白(MBP)、突觸核蛋白、熱休克蛋白beta-1、胰蛋白激酶A、磷酸酪氨酸蛋白磷酸酶、蘋(píng)果酸脫氫酶、精氨酸激酶、一型細(xì)胞骨架蛋白、Vesicle amine transport protein1、塔爾羊蛋白5、電子傳遞黃素蛋白；上調(diào)調(diào)蛋白1個(gè)：神經(jīng)絲蛋白。 結(jié)論：1000mg/kg TOCP暴露和40mg/kg PMSF前干預(yù)可獲得穩(wěn)定的雞OPIDN模型和PMSF干預(yù)模型。通過(guò)雙向電泳和質(zhì)譜分析技術(shù)從雞脊髓組織中篩選和鑒定了14個(gè)可能與OPIDN密切相關(guān)蛋白。其中下調(diào)蛋白包括5個(gè)神經(jīng)組織相關(guān)蛋白，4個(gè)蛋白酶類蛋白和4個(gè)其他相關(guān)蛋白。上調(diào)蛋白1個(gè)，為神經(jīng)絲蛋白。
[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 鹵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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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