亚洲带绦虫实验感染乳猪肝脏的蛋白质组研究
发布时间：2018-11-26 11:06
【摘要】：目的:采用蛋白质组学技术和方法研究亚洲带绦虫实验感染乳猪后15 d、75 d幼虫寄生处肝脏组织与对照组肝组织的蛋白质表达差异,为进一步研究亚洲带绦虫感染致乳猪肝损伤的分子机制提供基础资料。方法:1.绦虫标本采集及虫卵收集:将采自贵州省都匀市良亩乡的亚洲带绦虫孕节解剖后,以生理盐水反复清洗并离心收集虫卵。2.乳猪实验动物模型的建立及肝脏样本采集:20日龄约克施格杂交乳猪12头随机分为实验组和对照组各6头,实验组以定量虫卵15万个/头灌胃感染。于感染后第15 d和第75 d解剖乳猪,取实验组囊尾蚴寄生处肝组织和对照组对应部位肝组织。3.iTRAQ技术检测分析实验组和对照组乳猪肝脏的差异蛋白表达:主要步骤:蛋白质制备、酶解和肽段定量、肽段标记、液相色谱-串联质谱分析、Mascot软件查库鉴定及定量分析和生物信息学分析。4.部分差异蛋白的验证:运用实时荧光定量PCR、免疫印迹方法和免疫组织化学技术对部分差异蛋白进行进一步验证。结果:1.根据一般形态学特征和分子生物学鉴定结果,证实2条都匀采集的带绦虫为亚洲带绦虫。2.实验组6头乳猪均感染了亚洲带绦虫囊尾蚴,感染率为100%。囊尾蚴只存在于乳猪肝脏,其它组织未检获。3.iTRAQ分析结果显示感染后第15 d实验组与对照组比较共有187个蛋白质(92个未知)有显著差异;感染后第75 d实验组与对照组比较共有158个蛋白质(72个未知)有显著差异。4.验证结果显示感染后第15 d,实验组半胱氨酸天冬氨酸蛋白酶3(Cysteine aspartyl proteinase 3,Caspase-3)mRNA和蛋白表达水平均低于对照组(P0.05,P0.01),感染后第75 d,实验组Caspase-3 mRNA和蛋白表达水平较对照组无明显变化(P0.05);感染后第15 d和75 d,实验组膜联蛋白A5(Annexin A5,Anxa5)和甲硫氨酰氨肽酶2(Methionine aminopeptidase 2,Metap2)mRNA和蛋白表达水平均高于对照组(P0.05,P0.01),而实验组细胞色素P4501A1(CytochromeP450 1A1,Cyp1a1)mRNA和蛋白表达水平均低于对照组(P0.05,P0.01);免疫组化结果显示Caspase-3、Anxa5阳性染色主要定位在肝细胞胞质中,呈黄色或棕黄色。结论:1.iTRAQ技术检测到了亚洲带绦虫实验感染乳猪肝脏的大量差异蛋白表达,为进一步研究亚洲带绦虫致中间宿主乳猪肝损伤的分子机制提供了基础资料。2.目标蛋白的验证结果提示Caspase-3可能参与了亚洲带绦虫感染乳猪15 d肝损伤的调节,而Cyp1a1、Anxa5和Metap2可能参与了亚洲带绦虫感染乳猪15 d和75 d肝损伤的调节。
[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.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R383.3
本文编号：2358390
[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.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R383.3
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