【作者】 张彧；

【导师】 李强； 李锦成；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2009， 博士

【摘要】 研究背景：近年来，胰腺癌发病率在全国乃至全世界均呈明显的上升趋势。大约40％的胰腺癌患者在诊断时就已经发生转移，而神经浸润转移是其最常见的转移途径和重要特点之一，也是导致胰腺癌病人术后复发率高和预后差的重要原因之一。目前认为神经浸润是引起胰腺癌复发和癌性疼痛的重要原因，是其独立预后指标。近年来，胰腺癌的侵袭与转移，尤其是胰腺癌的神经定向转移问题，日益成为临床与基础研究中的重点和热点。自从1889年Peget首次提出癌转移的“种子与土壤”学说、1929年Ewing提出肿瘤转移的“机械与解剖”学说，多强调癌细胞自身的侵袭能力、运动能力等生物学特性的改变以及对组织、器官的被动适应。多年来，从亚细胞、细胞及其微环境、细胞与宿主机体的相互关系等诸多方面进行了深入广泛的研究，初步形成现代肿瘤转移理论：基因参与下的转移调控机制。该机制认为，肿瘤是由诸多因素决定的多步骤、序贯性的生物学过程。除强调肿瘤细胞自身因素外，更强调促转移因素与抑转移因素间网络调节的平衡性、宿主微环境的可塑性、宿主整体／局部免疫和肿瘤细胞的主动适应性变化等。但胰腺癌神经组织浸润的确切分子机制至今尚不明朗。以往研究认为，主要是神经周围间隙中张力较低，易于肿瘤生长，胰腺癌神经浸润是在平面最小阻力处即神经束膜的薄弱部位侵入神经，并在神经周围间隙内连续浸润，到达远处形成新的转移灶。近年来的研究表明，某些表面特性分子和神经组织周围微环境成分在肿瘤的嗜神经性上可能起着重要作用。膜结构伸展刺突蛋白moesin，是ERM蛋白家族的成员之一，表达于许多细胞膜突出部位，如伪足、微绒毛等，对维持细胞骨架及运动都起着重要作用，参与人体生理状态下的许多基本生命活动，是近年来被广泛关注的一类特殊蛋白质。近年发现其广泛表达于多种肿瘤组织中，在肿瘤发生、浸润及转移过程中可能扮演着重要角色，这一观点近年来已在口腔鳞状上皮细胞癌、宫颈癌、头颈恶性肿瘤以及某些消化系统的恶性肿瘤组织中得到了证实。但moesin是否在胰腺癌转移途径中发挥了作用，其作用机制仍不太清楚。为此，本课题选择moesin——这一新近受到关注的指标作为研究切入点，探讨胰腺癌发生神经侵袭转移的原因和机制。目的：收集天津医科大学附属肿瘤医院病理科手术切除的胰腺癌组织标本，从免疫组织化学、RT-PCR、实时荧光定量PCR、ELISA等方法检测moesin、β-NGF和MMP-7在人胰腺癌组织中的表达分布，初步探讨moesin、β-NGF和MMP-7在人胰腺癌组织中的作用及其与临床病理学特征之间的关系。并在以上研究的基础上结合患者临床症状，探讨moesin、β-NGF和MMP-7与胰腺癌患者疼痛症状的关系，以及胰腺癌疼痛与其临床病理学改变的相关性，进一步明确其神经性疼痛产生的原因，并探索缓解胰腺癌患者疼痛的方法，提高其生存质量。方法：(1)采用免疫组织化学的方法检测胰腺癌组织中moesin、β-NGF和MMP-7各指标的表达、分布特点，并且分析其与临床病理学特征之间的关系。(2)应用PT-PCR、实时荧光定量PCR、ELISA等分子生物学技术检测该组标本中moesin、β-NGF和MMP-7的蛋白表达，在验证免疫组化结果的同时，分析moesin对癌细胞中β-NGF和MMP-7表达的影响，以探索moesin对胰腺癌细胞侵袭转移作用的分子机制和此过程中moesin可能存在的调控机制。(3)在上述二部分的基础上，初步探讨moesin、β-NGF和MMP-7与胰腺癌患者疼痛症状的关系，以及胰腺癌疼痛与其临床病理学改变的相关性，进一步明确其神经性疼痛产生的原因，并探索缓解胰腺癌患者疼痛的方法。(4)采用SPSS15．0统计学软件包进行数据处理，以p＜0．05为有显著性差异。结果：(1)moesin、β-NGF、MMP-7均参与胰腺癌的发生发展。44例胰腺癌组织中有24例存在神经浸润(几率为54．55％)。侵入型8例，围神经型16例。moesin的表达在胰腺癌组织中明显高于正常组织，在胰体尾部癌组织中高于胰头癌，在有神经侵犯组明显高于无神经侵犯组，而且靠近神经组织的癌细胞moesin阳性程度高于远离神经组织的癌细胞。此外，随着癌细胞分化程度的降低和临床TNM分期的增加，moesin表达水平增加，moesin表达在有淋巴结转移组比无淋巴结转移组明显增加。β-NGF、MMP-7的表达与胰腺癌临床病理学特征的关系和moesin相似，经过相关性分析显示β-NGF、MMP-7和moesin的表达呈现正相关。(2)运用RT-PCR、实时荧光定量PCR、ELISA等分子生物学检测方法从RNA转录、蛋白表达、基因表达等三个方面来验证胰腺癌细胞中moesin的表达情况。moesin在胰腺癌组织中高表达，且与β-NGF和MMP-7之间呈现正相关的表达，该结论与第一部分免疫组织化学方法得到的结论一致，从而进一步验证了moesin作为一个新近受到关注的指标与胰腺癌病情进展相关，并参与了胰腺癌的浸润转移机制。(3)胰腺癌伴有疼痛者比不伴有疼痛者的moesin、3-NGF和MMP-7水平高，而且第一和第二部分结果证明moesin、β-NGF和MMP-7水平与胰腺癌嗜神经性有关，说明胰腺癌患者的疼痛发生和胰腺癌的嗜神经性、胰腺癌细胞的moesin、β-NGF和MMP-7表达水平有关。胰腺癌肿瘤位置、大小、TNM分期以及其对胰内神经、胰腺前方被膜和腹膜后组织的浸润与疼痛有显著相关性，而分化程度、淋巴结转移以及其对胰外神经、远端胆管和十二指肠壁的浸润与疼痛没有显著相关性。结论：(1)moesin、β-NGF、MMP-7均参与胰腺癌的发生发展，与胰腺癌嗜神经性成正相关，并且与临床病理学特征尤其与肿瘤发生部位、神经浸润、淋巴结转移、癌细胞低分化和临床分期密切相关，同时与胰腺癌患者的疼痛关系密切。(2)从胰腺癌大体水平、分子生物学水平和患者临床症状等方面综合探讨了moesin参与胰腺癌嗜神经性生长的机理。更多还原

【Abstract】 Background:Pancreatic carcinoma(PAC) is common malignant neoplasms ofdigestive system tumors.Perineural invasion is one of the important features forpatients with pancreatic carcinoma,and it thus cause the difficulity in curativeresection,high local recurrence and abdominal pain.Perineural invasion has becomethe research emphasis and focus on pancreatic carcinoma.The“seed and soil”theoryforwarded by Peget in 1889 and“machine and atomy”theory forwarded by Ewing in1929 had almost emphasized tumor cell’ s progressiveness and invasion of itself,passive adaptability to tissue microenviroment,tissue and organ’ s anatomicalfactors abouts tumor metastasis and ignored the active contributions of organism.Atpresent,through more than 100 year’ s research,plentiful and substantial outcomeshave been made in the many levels and fields such as oncogene,biomacromolecule,subcell,cell,mesenchyma microenviroment and so on.On this basis,the modemtumor metastasis theory is formed gradually that tumor metastasis is a multisteps andsequential pathological process and emphasizes the balance between numerousmetastasis and antimetastasis factors,active plasticity and specificity of host targetorgan microenviroment and the whole and/or local immunity of the host.It wasbelieved that the tumor invasion appeared to follow the plane of least resistance of theperineural space,but recent reaearch shows that some molecules on the surface ofcancer cells may have effects on the progression of tumor perineural invasion.It isnecessary,therefore,to investigate the molecular mechanisms in patients withperineural invasion.Actin cytoskeleton reorganization is reportedly regulated byezrir/radixir/moesin(ERM) family.Moesin,one member of ERM family,mainlylocalizes on membrane structures such as microvillus or lamellipodia which are richin actin fibers.The functions of moesin proteins are regulated by its conformationalcharges:the intramolecular interaction between the N-and C-terminal domains of moesin protein charges masks several binding sites,leading to a dormant protein.When stimulated by extracellular signals,moesin protein changes its conformationand is activated as the bridge molecule crosslinking the actin with plasma membraneand initiating the reorganization of actin cytosleleton.In this manner,it regulates theprocess of cell growth and migration.Recently it has been reported that moesinprotein play important roles in several tumor development and metastasis.However,to the best of our knowledge,till now there is no research focused on the relevancebetween moesin protein and pancreatic carcinoma,in particular,the role andmechanism of moesin protein on pancreatic carcinoma metastasis.Objective:To study the moesin protein’s effects on the mechanism of pancreaticcarcinoma perineural invasion from operation tissue specimens.And to explore therelationship between moesin protein and the pain of the patients with pancreaticcarcinoma.Our study may provide in the future the novel therapeutic tools for thetreatment of PAC patients.Methods:(1) Expression and distribution of moesin,β-NGF and MMP-7 were detected inopration tissue specimens of pancreatic carcinoma with immunohistochemistry,RT-PCR,Real-time PCR and ELISA.The relations of moesin withβ-NGF andMMP-7 were analyzed.(2) Exploration the relationship between moesin,β-NGF and MMP-7 and thepain of the patients with pancreatic carcinoma.(3) All data were processed by SPSS version 15.0 analysis software.Results:(1) By immunohistochemistry,the expression of moesin,β-NGF and MMP-7 inthe well-,medium-and poor-differentiated PAC groups lie in the cytoplasm of tumorcell,higher than normal group (p＜0.05).The radio of PNI found was 54.55%.β-NGFand MMP-7 were correlated with perineural invasiveness.(2) By RT-PCR,real-time PCR and ELISA,we found that moesin,β-NGF andMMP-7 expression of PAC samples were higher than the other.There was great significantly difference between two groups.The result by molecular biologytechnology was correlated with it by immunohistochemistry.(3) We found that moesin,β-NGF and MMP-7 expression of in patients withpain were higher than those who without pain.Moesin,β-NGF and MMP-7 werecorrelated with perineural invasiveness.Conclusions:(1) Expression’s change of moesin,β-NGF and MMP-7 may induce the formof PAC.They may play an important role on perineural invasion of human pancreaticcarcinoma.(2) Moesin,β-NGF and MMP-7 expression have something to do with pain ofPAC patients.更多还原