胆道闭锁和特发性胆汁淤积症肝脏组织差异蛋白质组学研究
发布时间：2018-09-07 08:03
【摘要】：胆道闭锁及特发性胆汁淤积症均为新生儿期较为常见的肝胆系统疾病,其临床症状有许多共同之处,如黄疸均在新生儿及婴儿期出现,粪便均呈现淡黄色或白色,均存在肝脏增大及质地变硬；血液生化检查都存在高结合胆红素血症及酶学异常,以谷丙转氨酶、γ-GT等升高为主；另外,病理改变表现亦相似,均为肝细胞及毛细血管内胆汁淤积,汇管区炎性浸润、肝细胞变性坏死等,这些特征为临床诊断及鉴别诊断带来了很大困难。由于两种疾病的治疗方式及预后有显著差异,胆道闭锁需限期外科手术治疗,而新生儿肝内胆汁淤积症一般采取内科处理,因此,临床上迫切需要对这两种疾病进行谨慎鉴别。胆道闭锁发病机制未明,在病毒感染、免疫反应、发育异常及遗传因子等的参与下,发病过程涉及复杂的基因间和/或蛋白质间的相互作用,细胞内活动和环境的影响也会通过影响基因的表达及蛋白质的转录后加工。本研究采用双向电泳及相对和绝对定量同位素标记(iTRAQ)技术对胆道闭锁及新生儿肝内胆汁淤积症患者肝脏组织标本进行检测,并进行蛋白质表达谱差异的分析,从而筛选出差异蛋白,并对其中差异分子热休克蛋白90(HSP90)的差异表达进行验证,试图从蛋白质组学的研究角度对胆道闭锁发生机制及鉴别诊断提供线索。 第一部分双向电泳质谱技术对胆道闭锁与特发性胆汁郁积症肝组织的差异蛋白研究 目的 本研究分别选择胆道闭锁与特发性胆汁淤积症肝组织,从蛋白质层面入手,以双向凝胶电泳的方法对这两种疾病表达差异的蛋白进行筛选,并对其中的HSP90的定量表达进行验证,试图为疾病发生机制及鉴别诊断提供线索。 材料与方法 选取因梗阻性黄疸行腹腔镜下胆道造影术的患儿术中肝脏组织,术中明确诊断胆道闭锁者20例作为病例组,排除胆道闭锁者12例作为对照组,病例组内再分为2组各10例进行组内对照。提取肝脏组织总蛋白后组内蛋白等量混合,应用非线性PH3～10,长24cm的固定PH梯度的IPG胶条和12%的SDS-PAGE进行双向凝胶电泳分离总蛋白,考马斯亮蓝染色后用ImageScanner软件对凝胶进行扫描,PDquest8.0软件对两组的全蛋白质组表达谱进行差异分析,筛选两组间表达差异2倍以上的蛋白点。对胶中差异较大的18个蛋白点进行MALDI-TOF质谱分析,数据送入NCBI非冗余数据库,通过MASCOT搜索引擎检索。另选取了两组各13例患儿的肝脏组织,利用Western-Blot法对热休克蛋白90(HSP90)差异表达进行验证。 结果 1、在60个差异点中差异较大、在胶上显示清晰且与周围蛋白点分离较好的18个点进行MALDI-TOF质谱分析,鉴定出15个蛋白点,其中9个蛋白点在病例组中上调,包括肌球蛋白、RhoGDI、MnSOD、白蛋白、钙网蛋白以及波形蛋白；同时,6个蛋白点在对照组中上调,包括热休克蛋白、蛋白二硫键异构酶、氨甲酰磷酸合成酶以及膜粘连蛋白。 2、Westen blot检测病例组及对照组各13例患者肝组织中HSP90的表达,经Quantity One软件分析比较扫描图像的灰度值,病例组灰度平均值为53279±12288,对照组灰度平均值为276669±70025,P=0.030.05。 结论 双向凝胶电泳质谱发现肝组织内数种在胆道闭锁与胆汁淤积症之间差异表达的蛋白,这些差异蛋白可能与胆道闭锁及胆汁淤积症的发病、进展及临床转归有关,为探索胆道闭锁与胆汁淤积症的发病机制提供了一定的理论基础；HSP90定量结果显著升高,具有成为鉴别胆道闭锁与特发性胆汁淤积症的生物标志物之潜在价值。 第二部分ITRAQ技术对胆道闭锁与特发性胆汁淤积症肝组织的差异蛋白研究 目的 本研究采用iTRAQ技术对以上相同组织标本差异蛋白进行检测,以求获得更广的差异蛋白表达谱,研究相对和绝对定量同位素标记(iTRAQ)技术检测胆道闭锁与特发性胆汁郁积症肝组织的差异蛋白,为后续疾病发生机制及早期诊断、鉴别诊断等提供进一步研究线索。 材料和方法 肝组织样本来源同第一部分。在本部分研究中我们将对照组病例同样随机分为两组,即所有样本共分为病例组1、病例组2、对照组1和对照组2四组。病例组每组10例,对照组每组6例。采用iTRAQ定量技术联合液相色谱串联质谱(Liguid chromatography-tandem mass spectrometry,LC-MS/MS)技术,对两组患者的肝脏组织进行比较比较蛋白质组学研究。MS/MS数据采用Protein Pilot3.0软件进行肽段及蛋白质的鉴定及差异表达的定量搜库,鉴定蛋白所采用的置信区间大于95%,满足EF (error factor)2,且P0.05的条件,鉴定的蛋白具有统计学意义。鉴定到的蛋白质的定量结果经the Pro GroupTM Algorithm(Applied Biosystems)算法处理。组间表达差异大于20%即1.2倍或0.8倍即被认为该蛋白质表达存在差异。 结果 1、数据经搜库及数据处理后,共鉴定出iTRAQ标记定量信息的蛋白593个。设定差异蛋白的入选条件为通过同一蛋白在各组间表达量的比值,得到该蛋白在4组中表达量的相对值,并计算出在病例组和对照组组内表达平均值,将两组平均值相比后排序,选出比值最高和最低的蛋白分别25个,并排除其中组内差异组间差异的点,这样筛选出的蛋白共38个,其中21个在病例组上调,分别为SERPH、LV301、LV403、TBB2C、SSBP、DPY2、RL7、PRDX5、ECHD2、 THTM、KAD2、COMT、H2A2C、ACOT2、SRSF3、H15、IF4B、H2AZ、HNRPM、 MYH9以及THIM;17个在对照组上调,分别为PRDX1、HMCS2、TCPD、HBD、 GATM、THIK、FUCM、COF1、ACBP、GSTA2、CK054、HCDH、EST1、PRDX6、 TPIS、RLA2以及ASSY。 2、通过于双向电泳法的比对,发现热休克蛋白90、膜联蛋白A6、蛋白二硫键异构酶及肌球蛋白4个蛋白也在iTRAQ检测中被鉴定出来,且在两组病人中两种方法检测出的表达趋势一致。 结论 本次研究采用iTRAQ技术对胆道闭锁及特发性胆汁淤积症患者肝脏组织标本进行检测,发现差异蛋白,2D电泳技术发现的蛋白有所重叠,其互补使用使得本次研究发现的蛋白谱较广,得到较多的候选差异蛋白,为后续的筛选研究提供了更多的思路。
[Abstract]:Biliary atresia and idiopathic cholestasis are common hepatobiliary diseases in neonates. Their clinical symptoms have many similarities, such as jaundice in neonates and infants, pale yellow or white stool, liver enlargement and texture hardening, hyperconjugated bilirubinemia and hyperbilirubinemia in blood biochemical examination. In addition, the pathological changes were similar, including cholestasis in hepatocytes and capillaries, inflammatory infiltration in portal area, degeneration and necrosis of hepatocytes. These characteristics brought great difficulties for clinical diagnosis and differential diagnosis. The pathogenesis of biliary atresia is unknown. With the involvement of viral infection, immune response, abnormal development and genetic factors, the pathogenesis of biliary atresia is complicated. Inter-gene and/or protein interactions, intracellular activity and environmental effects also affect gene expression and post-transcriptional processing of proteins. In this study, two-dimensional electrophoresis and relative and absolute quantitative isotope labeling (iTRAQ) techniques were used to obtain liver samples from patients with biliary atresia and neonatal intrahepatic cholestasis. The differentially expressed proteins were screened by analyzing the differences in protein expression profiles, and the differential expression of heat shock protein 90 (HSP90) was verified to provide clues for the pathogenesis and differential diagnosis of biliary atresia from the perspective of proteomics.
Part I Differential Proteins in Liver Tissues of Biliary Atresia and Idiopathic Bile Stasis by Two-dimensional Electrophoresis-Mass Spectrometry
objective
In order to provide clues for the pathogenesis and differential diagnosis of biliary atresia and idiopathic cholestasis, two-dimensional gel electrophoresis (2-DE) was used to screen the differentially expressed proteins of biliary atresia and idiopathic cholestasis.
Materials and methods
Choose the liver tissue of the children with obstructive jaundice who underwent laparoscopic cholangiography, 20 cases of biliary atresia diagnosed definitely during the operation as the case group, 12 cases excluding biliary atresia as the control group, the case group was divided into two groups and 10 cases in each group for intra-group control. Two-dimensional gel electrophoresis (2-DE) was used to separate total proteins from IPG tapes with fixed PH gradient of 24 cm in length and 12% SDS-PAGE. ImageScanner software was used to scan the gels after staining with Coomassie brilliant blue. PDquest 8.0 software was used to analyze the difference of total proteomic expression profiles between the two groups and screen the protein spots with more than 2 times difference between the two groups. MALDI-TOF mass spectrometry was used to analyze 18 protein spots with significant difference, and the data were sent to NCBI non-redundant database and searched by MASCOT search engine.
Result
Fifteen protein spots were identified by MALDI-TOF mass spectrometry. Nine of them were up-regulated in the case group, including myosin, RhoGDI, MnSOD, albumin, calreticulin and vimentin. Increased levels of heat shock protein, protein disulfide isomerase, carbamoyl phosphate synthase, and membrane adhesion protein were observed in group A.
2. The expression of HSP90 was detected by Western blot in the liver tissues of 13 patients in the case group and 13 patients in the control group. The gray value of the scanned image was analyzed and compared by Quantity One software. The average gray value of the case group was 53279+12288, and that of the control group was 27669+70025, P=0.030.05.
conclusion
Two-dimensional gel electrophoresis mass spectrometry (2-DE-MS) showed that there were several differentially expressed proteins in liver tissue between biliary atresia and cholestasis. These differentially expressed proteins may be related to the pathogenesis, progression and clinical outcome of biliary atresia and cholestasis, which provides a theoretical basis for exploring the pathogenesis of biliary atresia and cholestasis. The results showed a marked increase in the dose, which may be a potential biomarker for differentiating between biliary atresia and idiopathic cholestasis.
The second part is the differential protein analysis of liver tissue between biliary atresia and idiopathic cholestasis by ITRAQ.
objective
In this study, iTRAQ technique was used to detect the differentially expressed proteins in the same tissues in order to obtain a wider spectrum of differentially expressed proteins. Relative and absolute quantitative isotope labeling (iTRAQ) technique was used to detect the differentially expressed proteins in the hepatic tissues of biliary atresia and idiopathic cholestasis. Break and provide further clues.
Materials and methods
In this part, we divided all the samples into four groups: case group 1, case group 2, control group 1 and control group 2. There were 10 cases in each group and 6 cases in each control group. MS / MS data were identified by Protein Pilot 3.0 software and quantitatively searched for protein fragments and differential expression. The confidence interval of the identified proteins was more than 95%, satisfying EF (error factor) 2 and P 0.05. Quantitative results of the identified proteins were processed by the Pro Group TM Algorithm (Applied Biosystems). Intergroup differences of more than 20%, i.e. 1.2 or 0.8 times, were considered to be differences in protein expression.
Result
1. After data collection and data processing, 593 proteins with quantitative information of iTRAQ markers were identified. The selected condition of differential proteins was to obtain the relative expression of the protein in the four groups by the ratio of the same protein expressed in each group, and calculate the average expression in the case group and the control group. The average expression of the two groups was phased. The highest and lowest ratios of 25 proteins were selected, and the differences between groups were excluded. Twenty-one proteins were up-regulated in the case group, including SERPH, LV301, LV403, TBB2C, SSBP, DPY2, RL7, PRDX5, ECHD2, THTM, KAD2, COMT, H2A2C, ACOT2, SRSF3, H15, IF4B, H2AZ, HNRPM, MYH9 and THIM. 17 were up-regulated in the control group, PRDX1, HMCS2, TCPD, HBD, GATM, THIK, FUCM, COF1, ACBP, GSTA2, CK054, HCDH, EST1, PRDX6, TPIS, RLA2 and ASSY.
2. Compared with two-dimensional electrophoresis, four proteins, heat shock protein 90, annexin A6, disulfide isomerase and myosin, were also identified in the iTRAQ assay, and the two methods showed the same expression trend.
conclusion
In this study, iTRAQ technique was used to detect the liver tissue samples of patients with biliary atresia and idiopathic cholestasis. Differential proteins were found, and the proteins found by 2D electrophoresis overlapped. The complementary use of iTRAQ technique made the protein spectrum found in this study wider, and more candidate differential proteins were obtained, which provided more information for the follow-up screening study. Many ideas.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R725.7
[Abstract]:Biliary atresia and idiopathic cholestasis are common hepatobiliary diseases in neonates. Their clinical symptoms have many similarities, such as jaundice in neonates and infants, pale yellow or white stool, liver enlargement and texture hardening, hyperconjugated bilirubinemia and hyperbilirubinemia in blood biochemical examination. In addition, the pathological changes were similar, including cholestasis in hepatocytes and capillaries, inflammatory infiltration in portal area, degeneration and necrosis of hepatocytes. These characteristics brought great difficulties for clinical diagnosis and differential diagnosis. The pathogenesis of biliary atresia is unknown. With the involvement of viral infection, immune response, abnormal development and genetic factors, the pathogenesis of biliary atresia is complicated. Inter-gene and/or protein interactions, intracellular activity and environmental effects also affect gene expression and post-transcriptional processing of proteins. In this study, two-dimensional electrophoresis and relative and absolute quantitative isotope labeling (iTRAQ) techniques were used to obtain liver samples from patients with biliary atresia and neonatal intrahepatic cholestasis. The differentially expressed proteins were screened by analyzing the differences in protein expression profiles, and the differential expression of heat shock protein 90 (HSP90) was verified to provide clues for the pathogenesis and differential diagnosis of biliary atresia from the perspective of proteomics.
Part I Differential Proteins in Liver Tissues of Biliary Atresia and Idiopathic Bile Stasis by Two-dimensional Electrophoresis-Mass Spectrometry
objective
In order to provide clues for the pathogenesis and differential diagnosis of biliary atresia and idiopathic cholestasis, two-dimensional gel electrophoresis (2-DE) was used to screen the differentially expressed proteins of biliary atresia and idiopathic cholestasis.
Materials and methods
Choose the liver tissue of the children with obstructive jaundice who underwent laparoscopic cholangiography, 20 cases of biliary atresia diagnosed definitely during the operation as the case group, 12 cases excluding biliary atresia as the control group, the case group was divided into two groups and 10 cases in each group for intra-group control. Two-dimensional gel electrophoresis (2-DE) was used to separate total proteins from IPG tapes with fixed PH gradient of 24 cm in length and 12% SDS-PAGE. ImageScanner software was used to scan the gels after staining with Coomassie brilliant blue. PDquest 8.0 software was used to analyze the difference of total proteomic expression profiles between the two groups and screen the protein spots with more than 2 times difference between the two groups. MALDI-TOF mass spectrometry was used to analyze 18 protein spots with significant difference, and the data were sent to NCBI non-redundant database and searched by MASCOT search engine.
Result
Fifteen protein spots were identified by MALDI-TOF mass spectrometry. Nine of them were up-regulated in the case group, including myosin, RhoGDI, MnSOD, albumin, calreticulin and vimentin. Increased levels of heat shock protein, protein disulfide isomerase, carbamoyl phosphate synthase, and membrane adhesion protein were observed in group A.
2. The expression of HSP90 was detected by Western blot in the liver tissues of 13 patients in the case group and 13 patients in the control group. The gray value of the scanned image was analyzed and compared by Quantity One software. The average gray value of the case group was 53279+12288, and that of the control group was 27669+70025, P=0.030.05.
conclusion
Two-dimensional gel electrophoresis mass spectrometry (2-DE-MS) showed that there were several differentially expressed proteins in liver tissue between biliary atresia and cholestasis. These differentially expressed proteins may be related to the pathogenesis, progression and clinical outcome of biliary atresia and cholestasis, which provides a theoretical basis for exploring the pathogenesis of biliary atresia and cholestasis. The results showed a marked increase in the dose, which may be a potential biomarker for differentiating between biliary atresia and idiopathic cholestasis.
The second part is the differential protein analysis of liver tissue between biliary atresia and idiopathic cholestasis by ITRAQ.
objective
In this study, iTRAQ technique was used to detect the differentially expressed proteins in the same tissues in order to obtain a wider spectrum of differentially expressed proteins. Relative and absolute quantitative isotope labeling (iTRAQ) technique was used to detect the differentially expressed proteins in the hepatic tissues of biliary atresia and idiopathic cholestasis. Break and provide further clues.
Materials and methods
In this part, we divided all the samples into four groups: case group 1, case group 2, control group 1 and control group 2. There were 10 cases in each group and 6 cases in each control group. MS / MS data were identified by Protein Pilot 3.0 software and quantitatively searched for protein fragments and differential expression. The confidence interval of the identified proteins was more than 95%, satisfying EF (error factor) 2 and P 0.05. Quantitative results of the identified proteins were processed by the Pro Group TM Algorithm (Applied Biosystems). Intergroup differences of more than 20%, i.e. 1.2 or 0.8 times, were considered to be differences in protein expression.
Result
1. After data collection and data processing, 593 proteins with quantitative information of iTRAQ markers were identified. The selected condition of differential proteins was to obtain the relative expression of the protein in the four groups by the ratio of the same protein expressed in each group, and calculate the average expression in the case group and the control group. The average expression of the two groups was phased. The highest and lowest ratios of 25 proteins were selected, and the differences between groups were excluded. Twenty-one proteins were up-regulated in the case group, including SERPH, LV301, LV403, TBB2C, SSBP, DPY2, RL7, PRDX5, ECHD2, THTM, KAD2, COMT, H2A2C, ACOT2, SRSF3, H15, IF4B, H2AZ, HNRPM, MYH9 and THIM. 17 were up-regulated in the control group, PRDX1, HMCS2, TCPD, HBD, GATM, THIK, FUCM, COF1, ACBP, GSTA2, CK054, HCDH, EST1, PRDX6, TPIS, RLA2 and ASSY.
2. Compared with two-dimensional electrophoresis, four proteins, heat shock protein 90, annexin A6, disulfide isomerase and myosin, were also identified in the iTRAQ assay, and the two methods showed the same expression trend.
conclusion
In this study, iTRAQ technique was used to detect the liver tissue samples of patients with biliary atresia and idiopathic cholestasis. Differential proteins were found, and the proteins found by 2D electrophoresis overlapped. The complementary use of iTRAQ technique made the protein spectrum found in this study wider, and more candidate differential proteins were obtained, which provided more information for the follow-up screening study. Many ideas.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R725.7
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