【作者】 王巍；

【导师】 戴显伟；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 博士

【摘要】 前言胰腺癌是一种恶性程度极高的肿瘤,一个重要原因是胰腺癌具有的高度侵袭转移特性。在前期研究中,利用仓鼠实验性胰腺癌模型建立了两种转移能力完全不同的细胞株,解离型高转移(PC-1.0)和非解离型低转移(PC-1)胰腺癌细胞。在前期研究中,我们通过cDNA微阵列分析(cDNA microarray analysis),发现核孔蛋白107(nucleoporin 107kDa, Nup107)及紧密连接蛋白claudin23是解离型高转移株PC-1.0细胞与非解离型低转移株PC-1细胞中的差异表达基因。核孔复合体(Nuclear pore complexes, NPC)是近年来发现的贯穿于核膜作为细胞核与细胞质物质交换的惟一通道,在细胞正常的生长与分化中发挥着重要作用。研究已发现核孔蛋白结构与功能的改变与疾病及肿瘤发生、发展的关系密切。Nup107是核孔复合体中的一员,是NPC结构的重要核孔蛋白。有文献报道脑恶性胶质瘤细胞中Nup107表达显著增多,但Nup107在胰腺癌中作用尚无报道,其在肿瘤细胞中的作用机制也不清楚。claudin蛋白是构成细胞膜紧密连接至关重要的成分,claudin属于一个多基因家族,到目前为止,已有24种claudin成员被发现,其遗传表达的变化可导致上皮细胞、内皮细胞结构破坏、功能受损,与多种疾病的发生、发展存在密切关系,现已证实claudin家族蛋白成员在肿瘤发生、发展,肿瘤侵袭转移中具有重要作用。有文献报道claudin23在肠型胃癌中下调,但其在胰腺癌中作用尚无报道,其在肿瘤细胞中的作用机制不清楚。前期研究表明促丝裂原活化蛋白激酶(MAPK)信号转导通路的活化密切参与胰腺发生发展过程的调控,其具体分支通路定位于表皮生长因子受体(EGFR)／丝裂原活化蛋白激酶激酶(MEK)／细胞外信号调节激酶(ERK)。但胰腺癌中Nup107及claudin23的表达与MAPK信号转导通路的关系尚不明确。本研究中利用免疫细胞化学和RNA干扰的等技术,探讨究Nup107及claudin23在胰腺癌细胞株中的作用及相关作用机制。初步探讨Nup107及claudin23在胰腺癌发生及进展中的作用。目的初步探讨Nup107及claudin23在胰腺癌进展中作用的分子机制。同时为开发新型抗胰腺癌侵袭转移的高效治疗方法提供具有重要参考价值的新靶标。方法1.用RT-PCR、Western-blot方法验证Nup107 mRNA在解离型高转移株PC-1.0细胞与非解离型低转移株PC-1细胞中表达存在差异。2.用脂质体转染法将Nup107 siRNA转染入解离型高转移株PC-1.0胰腺癌细胞。用RT-PCR和Western-blot观察转染48h后PC-1.0胰腺癌细胞中Nup107mRNA、蛋白的表达以及MEK mRNA、蛋白的表达变化；应用流式细胞技术及CCK-8法分别检测转染后PC-1.0胰腺癌细胞细胞周期及增殖的变化情况；Papanicolaou’S染色法观察细胞解离状态及Transwell小室结合Matrix胶检测侵袭能力的变化。3.用稳定转染MEK1-shRNA和MEK2-shRNA的PC-1.0细胞检测Nup107 mRNA及蛋白表达的变化。4.用RT-PCR、Western-blot及免疫细胞化学方法研claudin23的表达及定位变化与胰腺癌细胞解离的关系。结果解离型高转移株PC-1.0细胞中Nup107 mRNA及蛋白表达高于非解离型低转移株PC-1细胞中表达；转染Nup107 siRNA 48h组与各对照组细胞相比,Nup107的mRNA、蛋白的表达明显减少(P<0.01),PC-1.0细胞在转染48h后细胞Gl期比例增加,S期、G2期细胞减少,细胞增殖受到抑制；细胞解离受到抑制,侵袭能力降低。稳定转染MEK1-shRNA和MEK2-shRNA的PC-1.0细胞与PC-1.0细胞相比,在MEK1-shRNA细胞中nup107 mRNA及蛋白表达减少明显,而在MEK2-shRNA细胞中减少不明显。claudin23 mRNA及蛋白在解离型高转移株PC-1.0细胞中低表达,而非解离型低转移株PC-1细胞中高表达,MEK1-shRNA的PC-1.0细胞中claudin23 mRNA及蛋白表达增加,而在MEK2-shRNA细胞中变化不明显。而PC-1+DF处理后,claudin23表达明显减少(P<0.05)并破坏claudin23在细胞膜的定位,细胞开始解离。结论1.Nup107参与胰腺癌细胞解离状态、增殖、细胞周期及体外侵袭的调控。2.Nup107参与胰腺癌的发展过程,其具体的机制可能为主要通过MEK1信号转导通路调控胰腺癌的增殖及细胞周期的变化,并通过MEK2信号通路调控胰腺癌的癌细胞解离状态及侵袭能力。3. MEK1/2信号转导通路通过调控Nup107的表达,来影响胰腺癌细胞解离状态、增殖、细胞周期及侵袭能力。4.claudin23表达及定位与胰腺癌细胞解离状态密切相关,其可能的分子机制为,激活了MEK2信号通路。更多还原

【Abstract】 Pancreatic cancer is known to be an extremely lethal neoplasm, one of the reasons being that pancreatic cancer itself has an extremely high potential of invasion-metastasis. In our previous study, two pancreatic cancer cell lines with a different potential for invasion-metastasis, PC-1 with a low potential and PC-1.0 with a high potential of invasion-metastasis after intrapancreatic transplantation, were established in a Syrian golden hamster. In our previous study, Differential expression of candidate genes Nup107 and claudin23 were indentified by cDNA microarray analysis.The nuclear pore complex (NPC) is a massive,multiprotin structure responsible for traffic between the nucleus and cytoplasm, it plays a critical role,not only in cell survival,but also in other cellular events such as differentiation. It’s structure and function change is correlated to tumorigenesis and development. Nup107 is a component of the nuclear pore complex, a important nucleoporin.In the current study,the expression of Nup107 were increased in spongiocytoma cell.But it has no report about pancreatic cancer, and the mechanism remains incompletely understood.claudins are essential component of the intercellular tight junction, its are multigene family. Today, the claudin superfamily consists of 24 members in eukaryotes. the genetic expression change of claudin may induce endothelial cell and epithelial cell breakdown,and they are involved in many disease. The claudin family member may play an important role in tumorigenesis and development,also in invasion and metastasis. In the current study,the expression of claudin23 were decreased in intestinal type gastric cancer cell. But it has no report about signal transduction pathway, and the mechanism remains incompletely understood. In our previous study, the mitogen-activated protein kinase (MAPK) signal transduction pathway y plays a central role in pancreatic cancer progression. The special pathway align on epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). But the relationship between Nup107 or claudin23 and MAPK signal transduction pathway in pancreatic cancer is still unclear.In this study,intends to claudin23 by immunoocytochemistry and Nup107 through the expression of RNAi technology in the pancreatic cancer cell lines Preliminary study Nup107 and claudin23 in the pathogenesis of pancreatic cancer in the role in order to investigate the occurrence and development of pancreatic cancer relationshipObjectiveTo study the role of Nup107 and claudin23 in the pathogenesis of pancreatic cancer and molecule mechanisms; To provide a new targer to bring a new method of gene therapy for pancreatic cancer.MethodsUsing RT-PCR and Western-blot observed the mRNA and protein expression of Nup107 in PC-1 with a low potential and PC-1.0 with a high potential of invasion-metastasis. With Lipofectamine method Nup107 siRNA transfected into pancreatic cancer cells. Using RT-PCR and Western-blot observed the mRNA and protein expression of Nup107 and MEK in the PC-1.0 cells after transfection 48h and the flow cytometry, CCK-8, Papanicolaou’staining and a Matrix gel coated Tanswell plate were used separately to detect the cell cycle, changes in proliferation,cell dissociation and invasion ability. Using RT-PCR and Western-blot observed the mRNA and protein expression of Nup107 in the stable transfection MEKl-shRNA and MEK2-shRNA PC-1.0 cells. Using RT-PCR, Western-blot and immunoocytochemistry observed the mRNA and protein expression and distribution of claudin 23 ResultsThe mRNA and protein expression of Nup107 in PC-1.0 with a high potential of invasion-metastasis are higher than PC-1 with a low potential.Nup107 siRNA transfection group compared with the control group cells, Nup107’s mRNA, protein expression was significantly reduced (P<0.01). cells’proliferation was inhibited after transfection transfected,cells increased the proportion of G1 phase, S phase、G2 phase cells decreased and cell dissociation and invasion ability were also restrained. Nup107’s mRNA and protein expression was significantly reduced (P<0.01) in the stable transfection MEKl-shRNA PC-1.0 cells, but MEK2-shRNA PC-1.0 cells have no significant changes (P>0.05). The mRNA and protein expression of claudin23 in PC-1.0 with a high potential of invasion-metastasis are lower than PC-1 with a low potential (P<0.05). After incubation with dissocation factor conditioned medium(DF-CM) of PC-1.0 cells, plasma membrane distribution of claudin23 was obviously disrupted, and expressions of claudin23 decreased in PC-1 cells. claudin23 ’s mRNA and protein expression was significantly increased (P<0.05) in the stable transfection MEK1-shRNA PC-1.0 cells, but MEK2-shRNA PC-1.0 cells have no significant changes (P>0.05).ConclusionsNup107 has a key role of cell cycle, cell proliferation,cell dissociation and invasion ability in pancreatic cancer. Nup107 has important effect in the occurrence and development of pancreatic cancer cells. The effect of cell cycle and cell proliferation were through the cellular transduction MAPK/MEK1 pathway, The effect of cell dissociation and invasion ability were through the cellular transduction MAPK/MEK2 pathway. The effect of cell cycle, cell proliferation,cell dissociation and invasion ability were regulated the expression of Nup107 through the cellular transduction MAPK/MEK1/2 pathway. Arragement of expression and distribution of claudin23 is closely related to cell dissocation status in pancreatic cancer cell through MEK2 activation.更多还原