【作者】 张春燕；

【导师】 杨云生；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2009， 博士

【摘要】 [背景与目的]肠易激综合征(irritable bowel syndrome,IBS)是常见的功能性肠病,其发病机制尚不清楚。在前期工作中,本课题组应用蛋白质组学方法,通过双向凝胶电泳及质谱分析技术对IBS-D患者和正常人结肠黏膜蛋白进行对比研究,首次鉴定出烯醇化酶α(α-enolase)、醛缩酶A(aldolase A ALDOA)、ATP合酶d亚基(ATP synthase subunit d,ATP5H)、细胞质乙酰辅酶A乙酰基转移酶(CYtoSolic thiolase,CT)、硫氧还蛋白(thioredoxin,Trx)、醛脱氢酶2(aldehyde dehydrogenase 2,ALDH2)、异丁酰辅酶A脱氢酶(isobutyryl-CoA dehydro-genase,ACAD8)、WDR1蛋白(WD repeat-containing protein 1,WDR1)和T复合物蛋白1α亚单位(T-complex protein 1 subunitα,TCP-1α)等在IBS-D患者结肠表达显著异常。本研究应用免疫纽化、western blot、荧光定量PCR等方法以正常健康人为对照组,进一步验证以上蛋白在腹泻型IBS(diarrhea-predominant IBS,IBS-D)、便秘型(constipation-predominant,IBS-C)患者结肠黏膜的表达情况,同时检测三组结肠黏膜三磷酸腺苷(ATP)含量,旨在探讨IBS的发病机制的新机理并对未来研究提供新的思路。[材料与方法]IBS-D患者18例,IBS-C患者18例,同时符合功能性胃肠病罗马Ⅱ科研诊断标准和罗马Ⅲ诊断标准,健康志愿者18例为对照组。分别在进行结肠镜检查时,直视下于盲肠及乙状结肠粘膜各活检5块;4块立即置于冰盐水中洗净,液氮中保存;1块于福尔马林溶液中固定,石蜡包埋。应用Western blot和免疫组织化学等方法对以上蛋白质进行验证,应用荧光定量PCR观测硫氧还蛋白、烯醇化酶α、异丁酰辅酶A脱氢酶mRNA的水平。由于样本有限,所有样本分为a、b、c三组进行实验,各组样本数相同(IBS-D6例,IBS-C6例,健康对照组6例)。鉴定ALDOA、α-enolase与TCP-1α在结肠黏膜的表达选用a组样本进行实验;ACAT2、Trx与ACAD8应用b组样本;ATP5H、WDR1与ALDH2应用c组样本.选择同时符合罗马Ⅱ和罗马Ⅲ诊断标准的IBS-D7例,IBS-C组6例,健康对照组5例行高效液相色谱分析检测结肠黏膜组织ATP含量。[结果]α-enolase、ALDOA、Trx在IBS-D组乙状结肠、IBS-C组乙状结肠及IBS-D组盲肠表达量明显少于健康对照组的相应部位,差异显著。α-enolase mRNA在IBS-D盲肠的表达明显下调。Trx mRNA在IBS-D盲肠、IBS-C盲肠的表达与健康对照组相比明显下调;Trx mRNA的在IBS-D乙状结肠及IBS-C乙状结肠表达明显上调。CT在IBS-D盲肠、IBS-C组盲肠表达量高于健康对照组,差异显著。ACAD8、ATP5H在IBS-D组乙状结肠、IBS-C乙状结肠表达明显低于健康对照组;ACAD8 mRNA在IBS-D乙状结肠、IBS-C组乙状结肠的表达明显下调,差异显著。ALDH2在IBS-D组盲肠及IBS-C组盲肠表达量低于健康对照组,差异非常显著,ALDH2在IBS-C乙状结肠、IBS-D乙状结肠的表达明显高于健康对照组,差异显著。TCP-1α在IBS—C乙状结肠及IBS-D组乙状结肠、IBS-D组盲肠表达量低于健康对照组的相应部位,差异显著。WDR1在大肠黏膜表达并且在IBS-C组乙状结肠黏膜表达量高于健康对照组,差异显著。上述蛋白的异常表达均有统计学意义(P＜0.05或P＜0.01)。IBS-D乙状结肠、IBS-C乙状结肠的ATP含量明显低于健康对照组相应部位(P＜0.05);IBS-D盲肠、IBS-C盲肠的ATP含量虽然低于健康对照组,但无统计学意义。[结论]ATP5H、ACAD8、ALDH2等“线粒体酶”在IBS-D和(或)IBS-C表达异常提示IBS发病机制可能涉及线粒体分子或功能异常;α-enolase、ALDOA、ACAD8、CT、ALDH2、ACAD8的等代谢相关酶在IBS患者结肠的表达异常以及结肠ATP含量减少提示着IBS-D、IBS-C患者结肠存在能量代谢异常。TCP-1α、WDR1的表达异常又提示可能影响细胞的骨架、结构完整性或细胞的形态与运动等。以上差异蛋白在同组不同部位表达情况有时亦不一致。结肠的不同部位IBS病的分子机制可能并不相同。更多还原

【Abstract】 [Background and Aims]Irritable bowel syndrome(IBS) is one of the most common functional gastrointestinal disorders(FGIDs).Although IBS has been intensively investigated in recent years,its pathogenesis remains unexplained.The previous study from our group applied proteomics methods to screen and identify abnormal expression of proteins in IBS with diarrhea(IBS-D) by the two dimensional gel(2-DE) and the mass spectrometry.we first identified the nine abnoamal proteins,which include alpha-enolase(α-enolase),aldolase A (ALDOA),ATP synthase subunit d(ATP5H),cytosolic acetyl coA acetyltransferase(CT),thioredoxin(Trx) and aldehyde dehydrogenase 2 (ALDH2),isobutyryl-CoA dehydro-genase(ACAD8),WD repeat-containing protein 1(WDR1) and T-complex protein 1 subunit alpha(tcp-1α).In this study, Western blot,immunohistochemistry staining(IHC) and fluorescent quantitation polymerase chain reaction(QPCR) were performed to verify the proteins. ATP content was detected in colonic mucosa from the IBS-D group,IBS with constipation(IBS-C) group and normal control group.All these researchs may provide new clues to further research on the molecular mechisms of IBS.[Materials and Methods]Eighteen IBS-D patients,18 IBS-C patients fulfilling on the RomeⅡand RomeⅢcriteria and 18 healthy volunteers as normal control group were enrolled in our current study.Biopsies were taken from the mucosa of cecum and sigmoid colon through standard colonoscopy.Five pieces were obtained separately at each site.Four out of five pieces were processed by cold saline water,and frozen in liquid nitrogen immediately while the other one was fixed in formalin and then embedded in paraffin three days later. Western blot and IHC were also performed for further verification of the proteins. Fluorescent QPCR were carried out for observing the level ofα-enolase mRNA, ACAD8 mRNA,Trx mRNA.Due to the limited sample numbers,samples were divided into three groups(a,b,c).Each group included 6 IBS-D patients,6 IBS-C patients and 6 healthy volunteers.The "a" group was applied to identify the expression of ALDOA andα-enolase and TCP-1α.The " b " group was applied to identify the expression of ACAT2 and Trx and ACAD8.The "c" group was applied to identify the expression of ATP5H and WDR1 and ALDH2.We enrolled 7 IBS-D patients,6 IBS-C patients both fulfilling on the RomeⅡandⅢcriteria and 5 normal controls.ATP content in colonic mucosa was observed by high performance liquid chromatograph(HPLC).[Results]Western blot and IHC showed that the expression of ALDOA,α-enolase and Trx were markedly decreased in mucosa of cecum and sigmoid colon from IBS-D and IBS-C groups than that of normal controls.α-enolase mRNA was markedly downregulated in mucosa of cecum from IBS-D.Trx mRNA was downregulated in mucosa of cecum from IBS-D and IBS-C groups.Trx mRNA were significantly upregnlated in mucosa of sigmoid colon from IBS-D and IBS-C groups comparing with control subjects.The expression of CT was significantly upregulated in mucosa of cecum from IBS-D and IBS-C groups.ACAD8 and ATP5H were significantly downregulated in mucosa of sigmoid colon from IBS-D groups and IBS-C groups.ACAD8 mRNA was downregulated in sigmoid colon from IBS-D and IBS-C groups.ALDH2 was markedly lower in cecum from IBS-D and IBS-C groups.ALDH2 was markedly higher in sigmoid colon from IBS-D and IBS-C groups than control.TCP-1αwas markedly lower in cecum and sigmoid colon from IBS-D and IBS-C groups. WDR1 were statistically significantly higer in sigmoid colon from IBS-C group. There were statistical significance on abnormal expression of above-mentioned proteins from colonic mucosa(P＜0.05 or P＜0.01).ATP content was markedly lower in sigmoid colon from IBS-C group and IBS-D groups(p＜0.05). [Conclusion]ATP5H,ACAD8,ALDH2 belong to "mitochondrial enzymes" that are abnormal in colonic mucosa from IBS group.The results indicated that mitochondrial molecule or function defect may involve in the pathogenesis of IBS;α-enolase,ALDOA,ACAD8,CT,ALDH2,ACAD8 were metabolism enzymes.The abnormal expression of the enzymes and reduction of ATP content showed that colonic mucosa from IBS patients may involve in dysregulation of energy metabolism.The abnormal expression of TCP-1α, WDR1 may influence colonic cystoskeleton,cellularity,cellular morphology and cellular movement.The different proteins had different expression in different area from colon which suggests complicated pathogenesis of IBS.更多还原