超短肠综合征大鼠结肠代偿的蛋白质组学研究
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摘要
研究背景：短肠综合征(short bowel syndrome,SBS)是由于小肠广泛切除后引起的慢性吸收不良综合征，残存小肠<30cm时称为超短肠综合征（ultra-short bowelsyndrome,USBS）。USBS可引起严重的吸收障碍与营养不良，目前尚无满意的治疗手段，肠外营养（parenteral nutrition,PN）是其主要的治疗方法，USBS患者须终身依赖PN，给社会与家庭带来了沉重的负担。既往研究证明USBS时结肠也能发生代偿。如何促进结肠的代偿，增进结肠的吸收功能，是提高USBS患者生存质量的关键，也是USBS的治疗方向之一，但目前结肠代偿的发生机制尚未明确。我们前期研究证实了USBS大鼠结肠的超微结构和吸收功能均发生明显的代偿改变，同时，我们用蛋白质组学方法鉴定出蛋白质二硫键异构酶A3（PDIA3）、丙酮酸激酶（PK）等10种结肠代偿差异表达蛋白，进一步研究结肠代偿差异表达蛋白，推测可能的调控USBS结肠代偿的关健因素，对阐明结肠代偿的分子机制及筛选促结肠代偿的蛋白质分子具有重要意义，值得进一步研究。
目的：建立USBS肠内营养支持的大鼠模型，运用蛋白质组学技术研究其结肠代偿所表达的差异蛋白质，为阐明结肠代偿机制及研发促代偿药物提供实验依据。
方法：30只雄性SD大鼠随机分为三组：（1）超短肠组（n=10）：手术切除90%～95%小肠；（2）假手术组（n=10）：横断小肠，吻合后关腹。两组大鼠术后均给予肠内营养支持。（3）对照组(n=10)：正常大鼠用与上述两组相同的方法和营养液喂养。肠内营养支持21天后分别提取3组大鼠的结肠粘膜标本，应用双向凝胶电泳（two-dimensional electrophoresis,2-DE）、质谱分析（mass spectrometry,MS）等蛋白质组学技术分析3组大鼠结肠代偿的蛋白质并鉴定出差异蛋白质，通过查询相关生物信息数据库了解这些差异蛋白质的功能与分类，并初步分析其在结肠代偿中可能起的作用。
结果：1)应用2-DE技术得到三个实验组的3块凝胶图谱，选取表达差异均在2倍以上的蛋白质，进行比较、分析后得出：①超短肠组与正常组间有46个差异蛋白质点，其中表达上调的有41个点，下调的有5个点，仅出现在超短肠组的有35个点。②超短肠组与假手术组间有30个差异蛋白质点，其中表达上调的有20个点，下调的有10个点，仅出现在超短肠组的有6个点。③比较上述两组的差异蛋白质点，得到共同差异蛋白质点共8个，其中7个表达上调且大于2倍，1个表达下调且大于2倍，仅在超短肠组出现的有5个。2)选取其中4个表达上调的共同差异蛋白点进行MS，鉴定出这4种差异蛋白质为：乙醛脱氢酶2，主穹隆蛋白，Wnt2蛋白，Agr2蛋白。
结论：鉴定出4种与结肠代偿相关的差异蛋白质：乙醛脱氢酶2，主穹隆蛋白，Wnt2蛋白，Agr2蛋白，这些蛋白质可能在大鼠结肠代偿过程中的氧化还原反应，细胞生长分化、抗氧化应激、信号通路等环节发挥作用。
Background:Short bowel syndrome (SBS) is caused by chronic malabsorptionsyndrome due to extensive small bowel resection, when remnant small bowel (RSB)<30cm, is called ultra-short bowel syndrome (USBS).USBS may have a severemalabsorption and malnutrition, currently still did not have a satisfactory treatmentcurrently except parenteral nutrition (PN). Patients have to depend on PN for wholelifetimes, which lead heavy burden to the social and family. Some of the previousstudies had shown that when the USBS happened, the colon could also compensate.How to promote colon compensatory and enhance the absorption features of colon arethe key features to improve the quality of the USBS patients’ survive, and also is one ofthe treatment direction of USBS, but the mechanism of colon compensatory mechanismis still unclear. Our previous study confirmed the ultrastructure and the absorptionfeatures of the USBS rat colon had a obvious compensatory changes. By the time, wehave identified10kinds of associated colon compensatory protein such as proteindisulfide isomerase A3(PDIA3), pyruvate kinase (PK) with Proteomics. By furtherstudying the colon compensatory differences in protein expressions, and suggesting thepossible regulation of the USBS colon compensatory protein, so as to elucidate themolecular mechanisms of colon compensatory and presume the colon compensatorypromote molecules of have a great significance value, and it’s worthy for further study.
AIM: To establish a rat model of ultra-short bowel syndrome with enteral nutritionsupport and use proteomics technology to study the differential protein expression incompensatory colon, so as to provide an experiment basis to clarify the coloncompensatory mechanisms to support the research on compensatory drugs development.
Methods:30male SD rats, weighing250-300g, were randomly divided into threegroups:(1) Ultra-short bowel group (n=10): surgical removal of90%to95%of thesmall intestine;(2) Sham operation group (n=10): about12cm from the ileocecal valveof the small intestine underwent transection and anastomosis. The two groups of rats were fed by enteral nutrition.(3) Control group (n=10): normal rats with the samemethod as the above two groups and nutrition feeding. Postoperative enteral nutrition for21days, the three groups of rat colonic mucosa epithelium was extracted as specimens.By using the application of two-dimensional gel electrophoresis (2-DE) massspectrometry (MS) proteomic analysis, protein expression in the three groups werecompared in the rat colon and to identify differentially expressed proteins, to understandthe function and classification of these proteins by querying the relevant biologicalinformation database, and preliminary analysis of the possible role in coloncompensatory.
Results:(1) We established3gel maps from three experimental groups by2-DEtechnology, and then selected the expression proteins which had more than two timesdifferences. By comparison and analysis:1) Between Ultra-short bowel group andNormal group, that had46differential protein spots, including41up-regulated spots,5down-regulated spots, with only35spots appeared in the ultra-short bowel group.2) InUltra-short bowel group and the Sham group, there were30differential protein spots,with the up-regulation of20spots and down-regulation of10spots, within them sixspots only appeared in the ultra-short bowel group.3) By Comparing the above two setsof expressed proteins spots, we differentiated totally eight common protein spots, ofwhich seven were up-regulated more than2times, one down-regulated greater than2times, only five in the ultra-short bowel group.(2) Four common up-regulated proteinspots were selected for the MS processing, and four kinds of difference proteins wereidentified: Aldehyde dehydrogenase-2、Major vault protein、Wnt2protein、Agr2protein.
Conclusion:We identified four proteins that played an important role in coloniccompensation: Aldehyde dehydrogenase-2、Major vault protein、Wnt2protein、Agr2protein. These proteins may take part in the oxidation reduction reactions, cell growthand differentiation, anti-oxidative stress, signaling pathways, etc. in the rats’compensatory colon.
目的：建立USBS肠内营养支持的大鼠模型，运用蛋白质组学技术研究其结肠代偿所表达的差异蛋白质，为阐明结肠代偿机制及研发促代偿药物提供实验依据。
方法：30只雄性SD大鼠随机分为三组：（1）超短肠组（n=10）：手术切除90%～95%小肠；（2）假手术组（n=10）：横断小肠，吻合后关腹。两组大鼠术后均给予肠内营养支持。（3）对照组(n=10)：正常大鼠用与上述两组相同的方法和营养液喂养。肠内营养支持21天后分别提取3组大鼠的结肠粘膜标本，应用双向凝胶电泳（two-dimensional electrophoresis,2-DE）、质谱分析（mass spectrometry,MS）等蛋白质组学技术分析3组大鼠结肠代偿的蛋白质并鉴定出差异蛋白质，通过查询相关生物信息数据库了解这些差异蛋白质的功能与分类，并初步分析其在结肠代偿中可能起的作用。
结果：1)应用2-DE技术得到三个实验组的3块凝胶图谱，选取表达差异均在2倍以上的蛋白质，进行比较、分析后得出：①超短肠组与正常组间有46个差异蛋白质点，其中表达上调的有41个点，下调的有5个点，仅出现在超短肠组的有35个点。②超短肠组与假手术组间有30个差异蛋白质点，其中表达上调的有20个点，下调的有10个点，仅出现在超短肠组的有6个点。③比较上述两组的差异蛋白质点，得到共同差异蛋白质点共8个，其中7个表达上调且大于2倍，1个表达下调且大于2倍，仅在超短肠组出现的有5个。2)选取其中4个表达上调的共同差异蛋白点进行MS，鉴定出这4种差异蛋白质为：乙醛脱氢酶2，主穹隆蛋白，Wnt2蛋白，Agr2蛋白。
结论：鉴定出4种与结肠代偿相关的差异蛋白质：乙醛脱氢酶2，主穹隆蛋白，Wnt2蛋白，Agr2蛋白，这些蛋白质可能在大鼠结肠代偿过程中的氧化还原反应，细胞生长分化、抗氧化应激、信号通路等环节发挥作用。
Background:Short bowel syndrome (SBS) is caused by chronic malabsorptionsyndrome due to extensive small bowel resection, when remnant small bowel (RSB)<30cm, is called ultra-short bowel syndrome (USBS).USBS may have a severemalabsorption and malnutrition, currently still did not have a satisfactory treatmentcurrently except parenteral nutrition (PN). Patients have to depend on PN for wholelifetimes, which lead heavy burden to the social and family. Some of the previousstudies had shown that when the USBS happened, the colon could also compensate.How to promote colon compensatory and enhance the absorption features of colon arethe key features to improve the quality of the USBS patients’ survive, and also is one ofthe treatment direction of USBS, but the mechanism of colon compensatory mechanismis still unclear. Our previous study confirmed the ultrastructure and the absorptionfeatures of the USBS rat colon had a obvious compensatory changes. By the time, wehave identified10kinds of associated colon compensatory protein such as proteindisulfide isomerase A3(PDIA3), pyruvate kinase (PK) with Proteomics. By furtherstudying the colon compensatory differences in protein expressions, and suggesting thepossible regulation of the USBS colon compensatory protein, so as to elucidate themolecular mechanisms of colon compensatory and presume the colon compensatorypromote molecules of have a great significance value, and it’s worthy for further study.
AIM: To establish a rat model of ultra-short bowel syndrome with enteral nutritionsupport and use proteomics technology to study the differential protein expression incompensatory colon, so as to provide an experiment basis to clarify the coloncompensatory mechanisms to support the research on compensatory drugs development.
Methods:30male SD rats, weighing250-300g, were randomly divided into threegroups:(1) Ultra-short bowel group (n=10): surgical removal of90%to95%of thesmall intestine;(2) Sham operation group (n=10): about12cm from the ileocecal valveof the small intestine underwent transection and anastomosis. The two groups of rats were fed by enteral nutrition.(3) Control group (n=10): normal rats with the samemethod as the above two groups and nutrition feeding. Postoperative enteral nutrition for21days, the three groups of rat colonic mucosa epithelium was extracted as specimens.By using the application of two-dimensional gel electrophoresis (2-DE) massspectrometry (MS) proteomic analysis, protein expression in the three groups werecompared in the rat colon and to identify differentially expressed proteins, to understandthe function and classification of these proteins by querying the relevant biologicalinformation database, and preliminary analysis of the possible role in coloncompensatory.
Results:(1) We established3gel maps from three experimental groups by2-DEtechnology, and then selected the expression proteins which had more than two timesdifferences. By comparison and analysis:1) Between Ultra-short bowel group andNormal group, that had46differential protein spots, including41up-regulated spots,5down-regulated spots, with only35spots appeared in the ultra-short bowel group.2) InUltra-short bowel group and the Sham group, there were30differential protein spots,with the up-regulation of20spots and down-regulation of10spots, within them sixspots only appeared in the ultra-short bowel group.3) By Comparing the above two setsof expressed proteins spots, we differentiated totally eight common protein spots, ofwhich seven were up-regulated more than2times, one down-regulated greater than2times, only five in the ultra-short bowel group.(2) Four common up-regulated proteinspots were selected for the MS processing, and four kinds of difference proteins wereidentified: Aldehyde dehydrogenase-2、Major vault protein、Wnt2protein、Agr2protein.
Conclusion:We identified four proteins that played an important role in coloniccompensation: Aldehyde dehydrogenase-2、Major vault protein、Wnt2protein、Agr2protein. These proteins may take part in the oxidation reduction reactions, cell growthand differentiation, anti-oxidative stress, signaling pathways, etc. in the rats’compensatory colon.
引文
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[2]S.Weih,M.kessler. Langenbeck`s Arch Surg[M],Springer-verlag.2011.
[3]Kocoshis SA,Beath SV,Booth IW,et al.Intestinal failure and small boweltransplantation,including clinical nutrition:working group report of the second Worldcongress of Pediatric Gastroenterology,Hepatolopy and Nutrition [J].Pediatr-Gastroe-nterol Nutr.2004,39(3):655-s661.
[4]秦环龙,吴肇汉.极短小肠综合征的残存肠道功能以及各种营养物的影响[J].肠外与肠内营养杂志,1997,4(4):232-236.
[5]许剑民,靳大勇,吴肇汉.结肠在短肠综合征中的代偿作用[J].中华外科杂志,2000,38(4):312-314.
[6]Dowling RH, Booth CC. Structural and functional changes following small intestinalresection in the rat. Clin Sci1967;32(1):139-49.
[7]吴国豪.短肠综合征病人残余肠道的代偿[J].中国实用外科杂志,2005,(11):655-657
[8]郭庆峰.全小肠切除对大鼠结肠粘膜超微结构和吸收功能的影响[D].暨南大学,2008.
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