【作者】 尉坤；

【导师】 温进坤；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:大肠癌是我国常见的消化道恶性肿瘤之一,目前发病率有逐年上升的趋势,虽然已经对其进行了广泛的研究,但其发生、发展以及侵袭和转移的机制尚不是十分明确。核因子κB ( nuclear factor-kappa B, NF-κB )作为一类具有多向性调节作用的转录调控因子,通过调控多种下游基因的转录表达,而参与炎症、肿瘤形成等多种病理生理过程。为了观察与大肠癌发生、发展有关的炎性因子的表达特点,以及与NF-κB水平的关系,本研究检测分析了28例大肠癌及相应正常组织标本,对比观察了NF-κB、环氧合酶-2 ( COX-2 )、细胞间粘附分子-1 ( ICAM-1 )、血管细胞粘附分子-1 ( VCAM-1 )、基质金属蛋白酶-9 ( MMP-9 )等与肿瘤进展和转移有关的炎性标志物在正常和大肠癌组织中的表达变化,探讨了其在大肠癌中的表达特点、相互关系及其与大肠癌进展的相关性,试图为大肠癌的临床治疗和预后评价提供有价值的参考指标。方法:1标本:选取河北医科大学第四临床医院外二科2006年12月~2007年6月间住院行外科手术治疗的大肠癌患者28例,分别取其肿瘤组织与相应正常组织。2 Western blot分析:根据所检测的指标不同,分别取相应量的蛋白提取液( NF-κB p65为60μg, I-κBα为110μg, COX-2为120μg, ICAM-1和VCAM-1均为110μg, MMP-9为70μg, SM22α为80μg ),按比例与5×SDS上样缓冲液混匀,煮沸后,上样,进行SDS-PAGE。电转移至PVDF膜上。将印迹膜置于封闭液中封闭,随后与相应的一抗及二抗反应,最后用化学发光法检测抗原抗体结合区带。用数码成像分析系统软件对结果进行定量分析。结果:1大肠癌组织中NF-κB及其抑制物I-κBα的表达与肿瘤进展分期的关系在所检测的28份标本中,NF-κB在肿瘤组织中的表达水平明显高于正常组织的标本所占比例为60.7 % ( 17/28 )。肿瘤组织中I-κBα蛋白表达水平低于正常组织的比例为53.6 % ( 15/28 )。对Western blot区带进行定量分析表明,( 1 )从大肠癌组织中检测到的NF-κB蛋白表达水平明显的高于正常组织( P<0.05 );且Dukes D期NF-κB表达量升高的幅度明显的高于Dukes A期,差异有显著性( P<0.05 )。这些结果表明,NF-κB水平升高与大肠癌的发生、发展以及侵袭和转移具有相关性。( 2 ) Western blot结果也显示,大肠癌组织中,I-κBα蛋白水平明显低于正常组织,且随着肿瘤Dukes分期的进展,I-κBα呈逐渐降低趋势,但差异无明显的统计学意义( P>0.05 )。2大肠癌组织中I-κB激酶( IKK )磷酸化水平升高Western blot分析显示,在所检测的大肠癌组织中p-IKK的水平明显高于相应的正常组织,且随着肿瘤Dukes分期的进展,呈逐渐升高的趋势,差异有显著的统计学意义( P<0.05 )。3大肠癌组织中COX-2表达增加在所检测的大肠癌组织中,COX-2蛋白表达量升高的标本占64.3 % ( 18/28 )。Western blot区带定量分析表明,大肠癌组织中COX-2蛋白表达水平明显高于正常组织,差异有显著的统计学意义( P<0.05 )。但随着肿瘤Dukes分期的进展,其蛋白表达量逐渐升高的趋势并不是很明显,差异无显著性。4大肠癌组织中转移相关蛋白ICAM-1、VCAM-1、MMP-9和SM22α蛋白的表达与正常组织不同Western blot区带定量分析表明,所检测的大肠癌标本中,ICAM-1的蛋白表达水平多数高于正常组织,并且随着肿瘤Dukes分期的进展,其表达水平呈逐渐升高的趋势。统计学显示,在正常与肿瘤组织中,ICAM-1的水平变化有显著性差异( P<0.05 )。然而,VCAM-1在正常组织中也有较高水平的表达,在部分大肠癌组织中有升高趋势,差异没有显著性( P>0.05 )。在所检测的大肠癌标本中,MMP-9蛋白表达升高的标本占检测样本的比例为67.9 % ( 19/28 )。Western blot区带定量分析表明,大肠癌组织中MMP-9蛋白表达水平明显高于正常组织,差异有显著性( P<0.05 )。其表达水平与ICAM-1的表达呈正相关关系。SM22α是一种MMP-9表达的负调控蛋白。本实验发现,大肠癌组织中SM22α的表达量明显少于正常组织,统计学上差异具有显著性( P<0.05 )。另外,SM22α蛋白的表达量随着MMP-9蛋白表达水平的升高而逐渐降低,两者呈负相关关系。结论:1大肠癌组织中NF-κB的表达水平明显高于正常组织,并且随着肿瘤Dukes分期的进展而呈逐渐增加的趋势。2大肠癌组织中I-κBα的活性与表达水平明显低于正常组织;其上游激酶p-IKK的水平则明显高于正常组织,表明IKK的磷酸化活化及I-κBα水平降低与大肠癌的进展密切相关。3在大肠癌组织中,与肿瘤进展和转移相关的蛋白COX-2、ICAM-1、VCAM-1和MMP-9的表达水平均明显高于正常组织,且与IKK磷酸化水平具有正相关关系。表明IKK的激活在大肠癌的发生发展中发挥关键作用。4大肠癌组织中,MMP-9表达的负调控蛋白SM22α水平是降低的,这可能是加速肿瘤进展的机制之一。更多还原

【Abstract】 Objective: Colorectal carcinoma is one of the most common malignant tumors in our country. The tendency of incidence is increasingly today. Despite its extensive research, the mechanism of development and metastasis of colorectal carcinoma is unclear. NF-κB, as a mediator of inflammation, is involved in tumor development processes.To investigate the expressive patterns of inflammatory factors, and the relationship between them and NF-κB expression during colorectal carcinoma development, the present study detected the expression of several inflammatory markers such as NF-κB, COX-2, ICAM-1, VCAM-1 and MMP-9, which are related to tumor progression and metastasis. The good of the study is to provide valuable reference for clinical treatment and prognosis.Methods:1 Specimens: 28 cases of colorectal carcinoma specimens and matched normal tissues were collected from patients operated in the Surgery Department of the Fourth Hospital of Hebei Medical University during December 2006 to June 2007. All specimens were determined by pathology.2 Western blot analyses: Equal amounts of protein extracts from normal and colorectal carcinoma tissues were separated on 8%SDS-PAGE, and then blotted onto PVDF membrane. The membrane was immunologically stained with specific antibodies. The results were analyzed by digital imaging system.Results:1 Relationship between the expression of NF-κB and I-κBαand tumor progression(1) The level of NF-κB in colorectal carcinoma tissues was higher than that in the normal tissues (P<0.05), and the NF-κB expression in Dukes D was significantly higher than that in Dukes A ( P<0.05 ). These results indicated that the increased NF-κB is positively correlated with the progression of colorectal carcinoma. (2) The level of I-κBαin colorectal carcinoma tissues was lower than that in the normal tissues, but the difference was not statistically significant (P>0.05).2 The level of phosphorylated-IKK was increased in colorectal carcinoma tissuesWestern blot analysis showed that the level of p-IKK in colorectal carcinoma tissues was obviously higher than that in the normal tissues, and with the progression of Dukes stage, its level was gradually increased. There was a significant difference (P<0.05).3 The level of COX-2 was increased in colorectal carcinoma tissuesWestern blot analysis showed that the level of COX-2 in colorectal carcinoma tissues was obviously higher than that in the normal tissues, there was a significant difference (P<0.05). However, with the progression of Dukes stage, COX-2 level was not changed (P>0.05).4 The level of metastasis-associated protein ICAM-1, VCAM-1, MMP-9 and SM22αin colorectal carcinoma tissues is different from that in normal tissuesWestern blot analysis showed that the level of ICAM-1 in colorectal carcinoma tissues was obviously higher than that in the normal tissues, and with the progression of Dukes stage, its level was gradually increased. There was a significant difference (P<0.05). However, the level of VCAM-1 expression was high in the normal tissues, and mildly increased in tumors (P>0.05).Western blot analysis showed that the level of MMP-9 in colorectal carcinoma tissues was obviously higher than that in the normal tissues, there was a significant difference (P<0.05). The level of MMP-9 is positively correlated with ICAM-1.In addition, we still found that the level of SM22αin colorectal carcinoma tissues was obviously lower than that in the normal tissues, there was a significant difference (P<0.05). The level of SM22αis negatively correlated with MMP-9.Conclusions:1 The expression level of NF-κB in colorectal carcinoma tissues was obviously higher than that in the normal tissues, and with the progression of Dukes stage, its level was gradually increased.2 The level of I-κBαin colorectal carcinoma tissues was lower than that in the normal tissues, while the level of p-IKK is obviously higher than that in the normal tissues. These indicated that the phosphorylation of IKK and degradation of I-κBαis related with the progression of colorectal carcinoma.3 The expression level of COX-2, ICAM-1, VCAM-1 and MMP-9 in colorectal carcinoma tissues is higher than that in normal tissues, and is positively corrected with the level of p-IKK. These indicated that the activation of IKK plays a key role in the development of colorectal carcinoma.4 The decreased SM22αmay be a mechanism resulting in tumor progression.更多还原