膽道閉鎖和特發(fā)性膽汁淤積癥肝臟組織差異蛋白質(zhì)組學(xué)研究
發(fā)布時(shí)間：2018-09-07 08:03
【摘要】：膽道閉鎖及特發(fā)性膽汁淤積癥均為新生兒期較為常見的肝膽系統(tǒng)疾病,其臨床癥狀有許多共同之處,如黃疸均在新生兒及嬰兒期出現(xiàn),糞便均呈現(xiàn)淡黃色或白色,均存在肝臟增大及質(zhì)地變硬；血液生化檢查都存在高結(jié)合膽紅素血癥及酶學(xué)異常,以谷丙轉(zhuǎn)氨酶、γ-GT等升高為主；另外,病理改變表現(xiàn)亦相似,均為肝細(xì)胞及毛細(xì)血管內(nèi)膽汁淤積,匯管區(qū)炎性浸潤(rùn)、肝細(xì)胞變性壞死等,這些特征為臨床診斷及鑒別診斷帶來(lái)了很大困難。由于兩種疾病的治療方式及預(yù)后有顯著差異,膽道閉鎖需限期外科手術(shù)治療,而新生兒肝內(nèi)膽汁淤積癥一般采取內(nèi)科處理,因此,臨床上迫切需要對(duì)這兩種疾病進(jìn)行謹(jǐn)慎鑒別。膽道閉鎖發(fā)病機(jī)制未明,在病毒感染、免疫反應(yīng)、發(fā)育異常及遺傳因子等的參與下,發(fā)病過程涉及復(fù)雜的基因間和/或蛋白質(zhì)間的相互作用,細(xì)胞內(nèi)活動(dòng)和環(huán)境的影響也會(huì)通過影響基因的表達(dá)及蛋白質(zhì)的轉(zhuǎn)錄后加工。本研究采用雙向電泳及相對(duì)和絕對(duì)定量同位素標(biāo)記(iTRAQ)技術(shù)對(duì)膽道閉鎖及新生兒肝內(nèi)膽汁淤積癥患者肝臟組織標(biāo)本進(jìn)行檢測(cè),并進(jìn)行蛋白質(zhì)表達(dá)譜差異的分析,從而篩選出差異蛋白,并對(duì)其中差異分子熱休克蛋白90(HSP90)的差異表達(dá)進(jìn)行驗(yàn)證,試圖從蛋白質(zhì)組學(xué)的研究角度對(duì)膽道閉鎖發(fā)生機(jī)制及鑒別診斷提供線索。 第一部分雙向電泳質(zhì)譜技術(shù)對(duì)膽道閉鎖與特發(fā)性膽汁郁積癥肝組織的差異蛋白研究 目的 本研究分別選擇膽道閉鎖與特發(fā)性膽汁淤積癥肝組織,從蛋白質(zhì)層面入手,以雙向凝膠電泳的方法對(duì)這兩種疾病表達(dá)差異的蛋白進(jìn)行篩選,并對(duì)其中的HSP90的定量表達(dá)進(jìn)行驗(yàn)證,試圖為疾病發(fā)生機(jī)制及鑒別診斷提供線索。 材料與方法 選取因梗阻性黃疸行腹腔鏡下膽道造影術(shù)的患兒術(shù)中肝臟組織,術(shù)中明確診斷膽道閉鎖者20例作為病例組,排除膽道閉鎖者12例作為對(duì)照組,病例組內(nèi)再分為2組各10例進(jìn)行組內(nèi)對(duì)照。提取肝臟組織總蛋白后組內(nèi)蛋白等量混合,應(yīng)用非線性PH3～10,長(zhǎng)24cm的固定PH梯度的IPG膠條和12%的SDS-PAGE進(jìn)行雙向凝膠電泳分離總蛋白,考馬斯亮藍(lán)染色后用ImageScanner軟件對(duì)凝膠進(jìn)行掃描,PDquest8.0軟件對(duì)兩組的全蛋白質(zhì)組表達(dá)譜進(jìn)行差異分析,篩選兩組間表達(dá)差異2倍以上的蛋白點(diǎn)。對(duì)膠中差異較大的18個(gè)蛋白點(diǎn)進(jìn)行MALDI-TOF質(zhì)譜分析,數(shù)據(jù)送入NCBI非冗余數(shù)據(jù)庫(kù),通過MASCOT搜索引擎檢索。另選取了兩組各13例患兒的肝臟組織,利用Western-Blot法對(duì)熱休克蛋白90(HSP90)差異表達(dá)進(jìn)行驗(yàn)證。 結(jié)果 1、在60個(gè)差異點(diǎn)中差異較大、在膠上顯示清晰且與周圍蛋白點(diǎn)分離較好的18個(gè)點(diǎn)進(jìn)行MALDI-TOF質(zhì)譜分析,鑒定出15個(gè)蛋白點(diǎn),其中9個(gè)蛋白點(diǎn)在病例組中上調(diào),包括肌球蛋白、RhoGDI、MnSOD、白蛋白、鈣網(wǎng)蛋白以及波形蛋白；同時(shí),6個(gè)蛋白點(diǎn)在對(duì)照組中上調(diào),包括熱休克蛋白、蛋白二硫鍵異構(gòu)酶、氨甲酰磷酸合成酶以及膜粘連蛋白。 2、Westen blot檢測(cè)病例組及對(duì)照組各13例患者肝組織中HSP90的表達(dá),經(jīng)Quantity One軟件分析比較掃描圖像的灰度值,病例組灰度平均值為53279±12288,對(duì)照組灰度平均值為276669±70025,P=0.030.05。 結(jié)論 雙向凝膠電泳質(zhì)譜發(fā)現(xiàn)肝組織內(nèi)數(shù)種在膽道閉鎖與膽汁淤積癥之間差異表達(dá)的蛋白,這些差異蛋白可能與膽道閉鎖及膽汁淤積癥的發(fā)病、進(jìn)展及臨床轉(zhuǎn)歸有關(guān),為探索膽道閉鎖與膽汁淤積癥的發(fā)病機(jī)制提供了一定的理論基礎(chǔ)；HSP90定量結(jié)果顯著升高,具有成為鑒別膽道閉鎖與特發(fā)性膽汁淤積癥的生物標(biāo)志物之潛在價(jià)值。 第二部分ITRAQ技術(shù)對(duì)膽道閉鎖與特發(fā)性膽汁淤積癥肝組織的差異蛋白研究 目的 本研究采用iTRAQ技術(shù)對(duì)以上相同組織標(biāo)本差異蛋白進(jìn)行檢測(cè),以求獲得更廣的差異蛋白表達(dá)譜,研究相對(duì)和絕對(duì)定量同位素標(biāo)記(iTRAQ)技術(shù)檢測(cè)膽道閉鎖與特發(fā)性膽汁郁積癥肝組織的差異蛋白,為后續(xù)疾病發(fā)生機(jī)制及早期診斷、鑒別診斷等提供進(jìn)一步研究線索。 材料和方法 肝組織樣本來(lái)源同第一部分。在本部分研究中我們將對(duì)照組病例同樣隨機(jī)分為兩組,即所有樣本共分為病例組1、病例組2、對(duì)照組1和對(duì)照組2四組。病例組每組10例,對(duì)照組每組6例。采用iTRAQ定量技術(shù)聯(lián)合液相色譜串聯(lián)質(zhì)譜(Liguid chromatography-tandem mass spectrometry,LC-MS/MS)技術(shù),對(duì)兩組患者的肝臟組織進(jìn)行比較比較蛋白質(zhì)組學(xué)研究。MS/MS數(shù)據(jù)采用Protein Pilot3.0軟件進(jìn)行肽段及蛋白質(zhì)的鑒定及差異表達(dá)的定量搜庫(kù),鑒定蛋白所采用的置信區(qū)間大于95%,滿足EF (error factor)2,且P0.05的條件,鑒定的蛋白具有統(tǒng)計(jì)學(xué)意義。鑒定到的蛋白質(zhì)的定量結(jié)果經(jīng)the Pro GroupTM Algorithm(Applied Biosystems)算法處理。組間表達(dá)差異大于20%即1.2倍或0.8倍即被認(rèn)為該蛋白質(zhì)表達(dá)存在差異。 結(jié)果 1、數(shù)據(jù)經(jīng)搜庫(kù)及數(shù)據(jù)處理后,共鑒定出iTRAQ標(biāo)記定量信息的蛋白593個(gè)。設(shè)定差異蛋白的入選條件為通過同一蛋白在各組間表達(dá)量的比值,得到該蛋白在4組中表達(dá)量的相對(duì)值,并計(jì)算出在病例組和對(duì)照組組內(nèi)表達(dá)平均值,將兩組平均值相比后排序,選出比值最高和最低的蛋白分別25個(gè),并排除其中組內(nèi)差異組間差異的點(diǎn),這樣篩選出的蛋白共38個(gè),其中21個(gè)在病例組上調(diào),分別為SERPH、LV301、LV403、TBB2C、SSBP、DPY2、RL7、PRDX5、ECHD2、 THTM、KAD2、COMT、H2A2C、ACOT2、SRSF3、H15、IF4B、H2AZ、HNRPM、 MYH9以及THIM;17個(gè)在對(duì)照組上調(diào),分別為PRDX1、HMCS2、TCPD、HBD、 GATM、THIK、FUCM、COF1、ACBP、GSTA2、CK054、HCDH、EST1、PRDX6、 TPIS、RLA2以及ASSY。 2、通過于雙向電泳法的比對(duì),發(fā)現(xiàn)熱休克蛋白90、膜聯(lián)蛋白A6、蛋白二硫鍵異構(gòu)酶及肌球蛋白4個(gè)蛋白也在iTRAQ檢測(cè)中被鑒定出來(lái),且在兩組病人中兩種方法檢測(cè)出的表達(dá)趨勢(shì)一致。 結(jié)論 本次研究采用iTRAQ技術(shù)對(duì)膽道閉鎖及特發(fā)性膽汁淤積癥患者肝臟組織標(biāo)本進(jìn)行檢測(cè),發(fā)現(xiàn)差異蛋白,2D電泳技術(shù)發(fā)現(xiàn)的蛋白有所重疊,其互補(bǔ)使用使得本次研究發(fā)現(xiàn)的蛋白譜較廣,得到較多的候選差異蛋白,為后續(xù)的篩選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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.7
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