【作者】 李明；

【导师】 陈主初；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 人类的结肠具有吸收和分泌的重要机能,参与机体对水、电解质平衡的调节,维持内环境稳定。结肠的上皮细胞是致病因子与外环境作用于机体的主要靶细胞之一,也是机体许多病理变化的始发环节及主要病灶所在。衰老(aging)通常被定义为机体的正常生理功能随着年龄的增长而缓慢减退的过程,它是一种正常的、复杂的、不可逆的生物学过程,是机体的功能发生衰退及生理功能紊乱的综合表现。结肠上皮同样会随着年龄的增长而出现生理功能逐渐减退,同时恶性肿瘤等多种肠道疾病的发病率明显升高。针对结肠上皮的衰老机制,国内外也已开展了许多相关的研究:Lopes等人观察到衰老大鼠的结肠平滑肌细胞线粒体的形态和功能都发生了改变;Ono等人发现,随着年龄的增加,消化道上皮的DNA突变频率增加,而以结肠的突变频率最高;Tsurudome等发现,在老年人的结肠癌组织中8-羟基脱氧鸟嘌呤(8-hydroxydeoxyguanosine)的水平升高,提示氧自由基对结肠上皮细胞DNA的损伤程度增加。以往的这些研究虽然揭示了一些结肠上皮衰老以及衰老结肠上皮对癌症易感的机理,但是这些研究多数是从某个或某几个基因出发,而结肠上皮的衰老是一个渐进的、多基因、多因素参与的复杂过程。另外,以往研究多集中在DNA和RNA水平,但由于基因转录和翻译水平的复杂性,DNA和RNA水平的变化并不能完全反映蛋白质水平的变化,而蛋白质是生理功能的执行分子,并且蛋白质的翻译后修饰状况、蛋白质的相互作用、蛋白质的动态变化等信息仅通过基因组研究是无法获知的。传统的对单个蛋白质的研究方式和研究技术已无法满足后基因组时代里对衰老这类复杂生命现象的研究要求,而以细胞内全部蛋白质为研究对象的蛋白质组学的出现则为衰老研究提供了新的研究思路和技术。差异表达蛋白质组学是蛋白质组学研究的一个重要研究内容,与试图了解细胞内所有的表达蛋白质—“表达蛋白质组学”不同的是,差异表达蛋白质组学研究更多的是关注于细胞在不同生理、病理状态下的差异表达蛋白质,以期寻找或发现不同生理或病理状态下的特征性蛋白质。为此,本研究以青年人和老年人的正常结肠粘膜上皮组织为研究对象,采用比较蛋白质组学技术和方法来研究人类结肠上皮衰老相关的蛋白质,为阐明结肠上皮衰老的分子机制提供有意义的线索。首先抽提制备青年组和老年组的结肠粘膜上皮组织的总蛋白质,再应用双向凝胶电泳技术建立了重复性和分辨率均较好的青年组和老年组结肠粘膜上皮组织总蛋白质的双向凝胶电泳图谱。经银染显色,ImageScanner扫描仪扫描凝胶,PDQuest图像分析软件对两组的组织总蛋白质的双向电泳图谱进行了比较,识别两组差异表达的蛋白质点47个,再联合采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾串联质谱(ESI-Q-TOF-MS)对差异表达的蛋白质进行分析,获取MALDI-TOF-MS肽质量指纹图谱及ESI-Q-TOF肽序列标签,再经Mascot软件搜索SWISS-PROT蛋白质数据库鉴定了其中的38个差异表达的蛋白质。为了验证比较蛋白质组学研究结果,采用Real-time quantitativeRT-PCR和Western blot分析以及免疫组织化学技术对部分差异表达蛋白质在青年组和老年组结肠粘膜上皮组织中的表达水平进行了检测,其结果与比较蛋白质组学研究的结果一致。为了进一步验证比较蛋白质组学筛选到的差异蛋白质是否为真正的与衰老相关的差异蛋白质,本文采用D-半乳糖诱导方法建立NIH/3T3细胞复制性衰老模型,应用Western blot检测2个差异蛋白质(EF-Tu、Rhodanese)在衰老NIH/3T3细胞中的表达变化,观察到EF-Tu和Rhodanese在衰老NIH/3T3细胞中的表达水平显著下调,说明EF-Tu和Rhodanese表达下调可能与体外细胞衰老有关。本研究在建立青年组和老年组结肠粘膜上皮组织总蛋白质双向电泳图谱的基础上,采用质谱分析结合数据库搜索共鉴定出38个差异表达蛋白质,这些蛋白质按功能主要可以分为九类,即抗氧化蛋白质、信号传导类蛋白质、与凋亡相关蛋白质、分子伴侣、与蛋白质折叠相关蛋白质、基本代谢相关的酶类、转录与翻译相关蛋白质、能量产生相关蛋白、细胞骨架蛋白。因此,本研究结果提示线粒体功能受损、抗氧化能力下降和细胞凋亡增加是结肠上皮衰老的主要原因,为阐明结肠上皮衰老的分子机制提供了有意义的新的线索。更多还原

【Abstract】 Human colon has the function of absorption and secretion,participates the regulation of water and electrolyte balance,and maintains the homeostasis. Colonic epithelial cell is one of the main target cells in the body,which is acted by causative agents and external environment.It is also the initiating link for many pathological changes and the location for most loci.Aging is usually defined as a process during which the normal physiological functions may gradually weaken along with the increase of age.It is a normal, complicated and irreversible biological process,and a comprehensive manifestation of the declined functions and physiological functional disorder in the body.Gradual declination of the physiological function may also occur to colonic epithelium along with the increase of age.Meanwhile,the occurrences of various intestinal diseases,such as malignant tumor,etc.is obviously increased.Aiming at the aging mechanism of colonic epithelium, many related studies have been carried out at home and abroad.Lopes,etc. observed the changes took place in the mitochondrial morphology and function of colonic smooth muscle cells in aged rats.Ono,etc.found,along with the increase of age,the DNA mutation frequency of epithelial cells in digestive tract was increased.In particular,the mutation frequency of colonic cells was the highest.Tsurudome,etc.found raised level of 8-hydroxydeoxyguanosine in colon carcinoma tissue of the aged,which suggested increased degree of injury on DNA of colonic epithelial cells by oxygen free radicals.Although previous studies have revealed the mechanisms of the ageing of the colonic epithelium and susceptibility of ageing colonic epithelium to cancer,most of these studies proceeded from certain or some genes.However, the ageing of colonic epithelium is a gradual complex process involved by multiple genes and multiple factors.Besides,these studies mainly centralized at DNA and RNA level.But due to the complexity of gene transcription and translation,changes in DNA and RNA cannot reflect the changes of protein completely.Besides,it is impossible to learn the modification information after the translation of protein,the interaction of protein,the dynamic changes of protein,etc.only by studies on genome.Traditional study and technology on a single protein cannot satisfy with the requirements of studying such complex vital phenomena as ageing in the postgenome times.But the emergence of proteome by taking intracellular entire protein as the object has provided new research train of thought and technology.Differential expressional proteome is an important part in proteome study.Different from expressional proteome(a method trying to understand whole intracellular expressional protein),study of differential expressional proteome pays more attention to differential expressional protein of cells under different physiological or pathological condition,thus to seek or find key protein under different physiological or pathological condition.So normal colonic mucous epithelial tissues of the young and the aged people were selected in this study to study protein which is related to the ageing of human colonic epithelium using comparative proteome technology and methods,which might provide significant clue for clarifying the molecular mechanism of ageing in the colonic epithelium.Firstly,comparative two-dimensional gel electrophoresis(2-DE) technology was performed to separate the total protein of colonic mucous epithelial tissues of the young and the aged people,respectively.The well-resolved,reproducible 2-DE pattems of the total protein of colonic mucous epithelial tissues of the young and the aged people were established. Then,PDQuest software was used to analyze 2-DE images and 47 differential expressional proteins between the two groups were discovered.These differential expressional proteins were analyzed by both peptide mass fingerprint(PMF)and peptide sequence tag(PST)based on MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry) and ESI-Q-TOF-MS(Electrospray ionization-quadrupole time of flight mass spectrometry).Among them,38 differential expressional proteins were identified through searching SWISS-PROT protein database with Mascot software.In order to verify the results of comparative proteome study,real-time quantitative RT-PCR、Western blot analysis and immunohistochemical staining were used to determine the differential expressional levels of the partial proteins of colonic mucosa epithelial tissues of the young group and the aged group,and the results were identical with the proteome analysis.In order to verify the results of comparative proteome study furtherly,a senescent model of NIH/3T3 cell was induced by D-gal,two proteins was analyzed by western-blot in the senescent cell.The result showed that the expresion of EF-Tu and Rhodanese was declined in the senescent cell.This suggested the alteration could be associated with cell senescence in vitro.Above all,38 differential expressional proteins between the two groups were identified by 2-DE in combination with MALDI-TOF-MS and ESI-Q-TOF-MS.These differential expressional proteins could be divided into nine main groups based on their functions:anti-oxidative proteins, proteins relative to signal transduction,proteins relative to apoptosis, chaperones,proteins relative to protein folding,metabolic enzymes,proteins relative to transcription and translation,proteins relative to energy production, cytoskeletal proteins.So the results of this study indicate that injury of mitochondrial function and decline of antioxidant capability may be important reasons for the aging of human colonic epithelium.These data provides useful new information for the study of the molecular mechanism of ageing of the colonic epithelium.更多还原