【摘要】：目的:采用蛋白質(zhì)組學(xué)技術(shù)和方法研究亞洲帶絳蟲(chóng)實(shí)驗(yàn)感染乳豬后15 d、75 d幼蟲(chóng)寄生處肝臟組織與對(duì)照組肝組織的蛋白質(zhì)表達(dá)差異,為進(jìn)一步研究亞洲帶絳蟲(chóng)感染致乳豬肝損傷的分子機(jī)制提供基礎(chǔ)資料。方法:1.絳蟲(chóng)標(biāo)本采集及蟲(chóng)卵收集:將采自貴州省都勻市良畝鄉(xiāng)的亞洲帶絳蟲(chóng)孕節(jié)解剖后,以生理鹽水反復(fù)清洗并離心收集蟲(chóng)卵。2.乳豬實(shí)驗(yàn)動(dòng)物模型的建立及肝臟樣本采集:20日齡約克施格雜交乳豬12頭隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組各6頭,實(shí)驗(yàn)組以定量蟲(chóng)卵15萬(wàn)個(gè)/頭灌胃感染。于感染后第15 d和第75 d解剖乳豬,取實(shí)驗(yàn)組囊尾蚴寄生處肝組織和對(duì)照組對(duì)應(yīng)部位肝組織。3.iTRAQ技術(shù)檢測(cè)分析實(shí)驗(yàn)組和對(duì)照組乳豬肝臟的差異蛋白表達(dá):主要步驟:蛋白質(zhì)制備、酶解和肽段定量、肽段標(biāo)記、液相色譜-串聯(lián)質(zhì)譜分析、Mascot軟件查庫(kù)鑒定及定量分析和生物信息學(xué)分析。4.部分差異蛋白的驗(yàn)證:運(yùn)用實(shí)時(shí)熒光定量PCR、免疫印跡方法和免疫組織化學(xué)技術(shù)對(duì)部分差異蛋白進(jìn)行進(jìn)一步驗(yàn)證。結(jié)果:1.根據(jù)一般形態(tài)學(xué)特征和分子生物學(xué)鑒定結(jié)果,證實(shí)2條都勻采集的帶絳蟲(chóng)為亞洲帶絳蟲(chóng)。2.實(shí)驗(yàn)組6頭乳豬均感染了亞洲帶絳蟲(chóng)囊尾蚴,感染率為100%。囊尾蚴只存在于乳豬肝臟,其它組織未檢獲。3.iTRAQ分析結(jié)果顯示感染后第15 d實(shí)驗(yàn)組與對(duì)照組比較共有187個(gè)蛋白質(zhì)(92個(gè)未知)有顯著差異;感染后第75 d實(shí)驗(yàn)組與對(duì)照組比較共有158個(gè)蛋白質(zhì)(72個(gè)未知)有顯著差異。4.驗(yàn)證結(jié)果顯示感染后第15 d,實(shí)驗(yàn)組半胱氨酸天冬氨酸蛋白酶3(Cysteine aspartyl proteinase 3,Caspase-3)mRNA和蛋白表達(dá)水平均低于對(duì)照組(P0.05,P0.01),感染后第75 d,實(shí)驗(yàn)組Caspase-3 mRNA和蛋白表達(dá)水平較對(duì)照組無(wú)明顯變化(P0.05);感染后第15 d和75 d,實(shí)驗(yàn)組膜聯(lián)蛋白A5(Annexin A5,Anxa5)和甲硫氨酰氨肽酶2(Methionine aminopeptidase 2,Metap2)mRNA和蛋白表達(dá)水平均高于對(duì)照組(P0.05,P0.01),而實(shí)驗(yàn)組細(xì)胞色素P4501A1(CytochromeP450 1A1,Cyp1a1)mRNA和蛋白表達(dá)水平均低于對(duì)照組(P0.05,P0.01);免疫組化結(jié)果顯示Caspase-3、Anxa5陽(yáng)性染色主要定位在肝細(xì)胞胞質(zhì)中,呈黃色或棕黃色。結(jié)論:1.iTRAQ技術(shù)檢測(cè)到了亞洲帶絳蟲(chóng)實(shí)驗(yàn)感染乳豬肝臟的大量差異蛋白表達(dá),為進(jìn)一步研究亞洲帶絳蟲(chóng)致中間宿主乳豬肝損傷的分子機(jī)制提供了基礎(chǔ)資料。2.目標(biāo)蛋白的驗(yàn)證結(jié)果提示Caspase-3可能參與了亞洲帶絳蟲(chóng)感染乳豬15 d肝損傷的調(diào)節(jié),而Cyp1a1、Anxa5和Metap2可能參與了亞洲帶絳蟲(chóng)感染乳豬15 d和75 d肝損傷的調(diào)節(jié)。
[Abstract]:Objective: to study the difference of protein expression in liver tissues of larva parasitic larva of Taenia asiatica infected suckling pigs on the 15th day after infected with Taenia asiatica by proteomics techniques and methods. To further study the molecular mechanism of liver injury caused by Taenia asiatica infection in suckling pigs. Methods: 1. Taenia tapeworm specimen collection and egg collection: collected from Duyun City, Guizhou Province Liangmu Township after the pregnancy of Taenia asiatica after dissection, washed with physiological saline repeatedly and centrifuged collection of eggs. 2. Establishment of experimental animal model and collection of liver samples in suckling pigs: twelve 20-day-old Yorkshire crossbred suckling pigs were randomly divided into experimental group (n = 6) and control group (n = 6). The experimental group was infused with 150 000 eggs per head by stomach. The suckling pigs were dissected on the 15th and 75th days after infection. 3.iTRAQ technique was used to detect and analyze the differential protein expression in the liver of suckling pigs in the experimental group and the control group. The main steps were protein preparation, enzymatic hydrolysis and peptide segment quantification, and peptide segment labeling. Liquid chromatography-tandem mass spectrometry, Mascot software library identification and quantitative analysis and bioinformatics analysis. 4. Verification of partial differential proteins: some differential proteins were further verified by real-time fluorescent quantitative PCR, Western blotting and immunohistochemistry. Results: 1. According to the general morphological characteristics and molecular biological identification results, it was confirmed that the two tapeworms collected by Duyun were Taenia asiatica. All of the 6 suckling pigs in the experimental group were infected with cysticercaria of Taenia asiatica and the infection rate was 100. Cysticercus cysticercus only existed in the liver of suckling pigs. 3.iTRAQ analysis showed that 187 proteins (92 unknown) were significantly different between the experimental group and the control group on the 15th day after infection. There were significant differences in 158 proteins (72 unknown) between the experimental group and the control group on the 75th day after infection. 4. The results showed that the expression levels of mRNA and protein of cysteine aspartate protease 3 (Cysteine aspartyl proteinase 3 (Caspase-3) in the experimental group were lower than those in the control group on the 15th day after infection (P0. 05, P0. 01), and 75 days after infection. The expression of Caspase-3 mRNA and protein in the experimental group was not significantly different from that in the control group (P0.05). On the 15th and 75th day after infection, the mRNA and protein expression levels of Annexin A5 (Annexin A5 Anxa5) and methionylaminopeptidase 2 (Methionine aminopeptidase 2 (Metap2) in the experimental group were higher than those in the control group (P0.05p0.01), while the expression of cytochrome P4501A1 (CytochromeP450 1A1) in the experimental group was higher than that in the control group (P0.01). The expression level of mRNA and protein in Cyp1a1 was lower than that in control group (P0.05, P0.01). Immunohistochemical results showed that Caspase-3,Anxa5 positive staining was mainly located in the cytoplasm of hepatocytes, yellow or brown. Conclusion: 1.iTRAQ technique can detect a large number of differentially expressed proteins in the liver of suckling pigs infected with Taenia asiatica, which provides basic data for further study on the molecular mechanism of liver injury induced by Taenia asiatica. 2. The results showed that Caspase-3 might be involved in the regulation of liver damage in pigs infected with Taenia asiatica on day 15, while Cyp1a1,Anxa5 and Metap2 might be involved in the regulation of liver damage in pigs infected with Taenia asiatica at day 15 and day 75.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R383.3

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