【作者】 殷涛；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 背景和目的胰腺癌是一种恶性的消化系统肿瘤,具有强烈的向周围组织侵袭的倾向以及对化疗的抵抗能力,导致胰腺癌病人的预后非常差。Snail是一种抑制型的转录因子,可以赋予上皮细胞移动以及抗凋亡的能力。其表达在多种肿瘤组织中都有报道。Snail可能在胰腺癌细胞中表达并且赋予胰腺癌细胞侵袭和抵抗化疗的能力。方法为了论证此假设,我们通过免疫组织化学的方法检测了Snail在胰腺癌组织中的表达,以及和临床病例指标的相关性。同时我们将Snail编码基因转染胰腺癌细胞系Panc-1细胞,通过Transwell侵袭小室法检测Snail对Panc-1细胞体外侵袭能力的影响;通过MTT细胞增殖试验检测转染Snail基因对Panc-1细胞增殖以及对5-氟尿嘧啶、吉西他滨等化疗药物敏感性的影响;通过裸鼠接种实验检测Snail基因对胰腺癌细胞体内生长的影响。结果Snail在胰腺癌组织中存在着表达,在56例胰腺癌组织中发现有20例Snail的表达呈阳性,阳性率达36%。此外Snail的表达同淋巴结转移、远处转移都存在有明显的相关性。Panc-1细胞在转染了Snail基因之后发生了明显的从上皮细胞表型向间叶细胞表型的转化,同时其在体内外的侵袭能力也大大增强。另外Snail的表达还赋予了Panc-1细胞对5-氟尿嘧啶和吉西他滨的抵抗能力。结论Snail在胰腺癌组织中存在着表达,并且可以赋予胰腺癌细胞高度的侵袭和抵抗凋亡的能力。Snail有可能作为一种预测胰腺癌恶性程度的标志,发展针对Snail的治疗措施可能为提高胰腺癌患者的预后提供新的思路。目的探讨上皮向间叶转化(EMT)在胰腺癌组织中的发生情况,研究上皮细胞标记物E-cadherin、间叶细胞标记物vimentin在胰腺癌组织及胰腺癌细胞中的表达情况以及与胰腺癌恶性生物学行为的相关性。方法采用免疫组织化学的方法检测56例胰腺癌组织中E-cadherin、vimentin蛋白的表达,并与临床病理资料作对照分析。同时应用Western blot和免疫荧光技术检测Snail、E-cadherin、vimentin三种标记物分子在不同胰腺癌细胞Miapaca-2、Panc-1、BxPc-3中的表达情况,通过Transwell侵袭实验检测不同细胞侵袭能力的差异。结果56例胰腺癌组织中26例(46.4%)观察到E-cadherin表达降低,同时6例观察到vimentin表达上调。E-cadherin的表达抑制和胰腺癌的淋巴结浸润(P=0.021),远处转移(P=0.001),病理分级(P=0.049)呈显著性相关。而6例vimentin表达上调的胰腺癌组织中有3例发生了远处转移。E-cadherin表达的降低和vimentin的高表达都与Snail的表达存在着明显的相关性。在高分化的胰腺癌细胞BxPc-3中,E-cadherin呈现高表达,未发现有vimentin和Snail的表达,相对应的细胞体外侵袭能力较差。在中低分化的胰腺癌细胞Panc-1、Miapaca-2中vimentin、Snail表达逐渐增强,E-cadherin表达明显降低,相对应的细胞在体外的侵袭能力则大大增强。结论胰腺癌的侵袭过程中存在着肿瘤细胞上皮细胞标记物的降低,同时间叶细胞标记物的增强,EMT可能参与了胰腺癌侵袭性生物学行为。Snail可能通过调控EMT从而导致了胰腺癌细胞侵袭性表型。目的探讨缺氧微环境对胰腺癌细胞上皮向间叶转化的诱导效应以及可能机制。方法在缺氧微环境下培养胰腺癌细胞Panc-1,Transwell侵袭小室对比检测细胞在缺氧微环境下侵袭能力的变化情况。通过Western blot、免疫荧光检测缺氧对Panc-1细胞上皮细胞标记分子E-cadherin、间叶细胞标记分子vimentin表达的影响。同时通过实时荧光定量PCR检测缺氧对Panc-1细胞Snail mRNA表达的影响。为了探讨缺氧微环境诱导胰腺癌细胞发生表型改变的机制,将编码HIF-1αsiRNA的真核表达载体pGenesil-1-HIF-1α及对照载体瞬时转染Panc-1细胞,通过缺氧处理,Western blot检测沉默HIF-1α之后对Panc-1细胞E-cadherin、vimentin表达的影响;逆转录PCR检测抑制HIF-1α对Snail mRNA表达的影响;通过Transwell侵袭小室检测对细胞体外侵袭能力的影响。结果处于缺氧微环境下的Panc-1胰腺癌细胞在体外侵袭能力大大增强,缺氧可以抑制上皮细胞标记物E-cadherin、增强间叶细胞标记分子vimentin的表达,活化转录因子Snail的表达。缺氧微环境下通过RNA干扰技术沉默HIF-1α之后可以恢复缺氧对E-cadherin的抑制,同时降低vimentin和Snail的表达,抑制缺氧环境中细胞在体外的侵袭能力。结论缺氧微环境可以活化HIF-1α的表达,而HIF-1α可能通过诱导Snail进而促进胰腺癌细胞发生上皮向间叶转化产生侵袭性表型。靶向HIF-1α、Snail通路的治疗措施可能为抑制胰腺癌侵袭性生物学行为提供新的方法。更多还原

【Abstract】 Background Pancreatic cancer is a dreadful malignancy. Because of its tendency to metastasis and its resistance to chemotherapy, the prognosis remains poor. Snail is a transcriptional factor which endows epithelial cells with migratory and anti-apoptotic abilities. Its expression has been demonstrated in many tumors. We hypothesized that Snail may be expressed in pancreatic cancer, and it may confer invasive and chemoresistant properties.Material, Methods, and results We immunohistochemically examined Snail expression in pancreatic cancer, and found that it was expressed in 20 of 56 (36%) samples of pancreatic cancer. The Snail expression had a close correlation with lymph node invasion and distant metastasis. After transfecting Snail cDNA into pancreatic cancer cell line Panc-1, we found that Snail triggered overt epithelial to mesenchymal transitions in Panc-1 cells. The tumor invasive ability in vitro was evaluated using a transwell invasive chamber. Snail dramatically promoted the invasive ability of Panc-1 cells. Chemosensitivity of Panc-1 cells to 5-fluorouracil or gemcitabine after Snail transfection was assayed by MTT cell proliferation assay. Overexpression of Snail enhanced the chemoresistance to 5-fluorouracil of gemcitabine at different dosages. Moreover, Snail transfected Panc-1 cells produced more spontaneous metastasis than parental untransfected cells after orthotopically injected into the pancreas of nude mice.Conclusion Snail is expressed in pancreatic cancer; it confers enhanced invasive ability and chemoresistance to pancreatic cancer cells. Snail may be a marker for predicting the malignancy of pancreatic cancer. Further therapy target to Snail may be of great benefit to pancreatic cancer patients. Objective To investigate the occurrence of EMT in pancreatic cancer and the expression of epithelial marker E-cadherin, mesenchymal marker vimentin in pancreatic cancer and its correlation with the malignant features.Methods Snail、E-cadherin、vimentin expression were determined in 56 cases of human pancreatic carcinoma with immunohistochemistry and the results were compared with pathology. Western blot and immunofluorescence were performed to compare the expression of E-cadherin, vimentin, Snail in different pancreatic cancer cells: Miapaca-2, Panc-1 and BxPc-3. Transwell invasion assay was performed to compare the in vitro invasive abilities of different cancer cells.Results Reduced expression of E-cadherin was found in 26 cases, vimentin, a mesenchymal marker, was found to be highly expressed in pancreatic cancer cells in 6 cases. Significant correlation was detected between the expression of Snail and the reduced expression of E-cadherin and the expression of vimentin. The expression of vimentin was high in Miapaca-2 pancreatic cancer cells and the E-cadherin was low, but the invasive ability was strong. There were no vimentin and Snail expression in BxPc-3 cells, but the E-cadherin expression was strong and the invasive ability was weak.Conclusion Represson of epithelial marker and accquistion of mesenchymal marker may be involved in the progression of pancreatic cancer. Re-expression of Snail in pancreatic cancer may accelerate invasion through epithelial to mesenchymal transition. Epithelial to mesenchymal transition may be mechanisms for pancreatic cancer cells to acquire the invasive ability. Objective To investigate whether hypoxia environment can induce epithelial to mesenchymal transition of pancreatic cancer cells and the potential mechanisms.Methods The pancreatic cancer cells Panc-1 was cultured in hypoxia environment. After cultured for indicated periods, the in vitro invasive ability of Panc-1 cells was compared with normoxia group using Transwell invasion assay. The epithelial marker E-cadherin and the mesenchymal marker vimentin were assayed by Western blot. The Snail mRNA was assayed by realtime PCR. To elucidate the potential mechanisms involved in the phenotypic changes of Panc-1 cells in hypoxia environment, the plasmid pGenesil-1-HIF-1αexpressing siRNA targeting at HIF-1αgene and the control plasmid was transient tansfected into the Panc-1 cells and were cultured for indicated times. Then, Western blot was performed to detect the changes of epithelial marker E-cadherin and the mesenchymal marker vimentin. The expression of Snail mRNA was detected by RT-PCR. The invasive abilities invtro was detected by Transwell invasion assay.Results The invasive ability was increased dramatically in hypoxia environment. Hypoxia repressed the expression of E-cadherin, and enhanced the vimentin expression. Besides, the Snail mRNA was increased in hypoxia environment. After silencing the expression of HIF-1αof Panc-1 cells in hypoxia environment, the expression of E-cadherin can be enhanced、the vimentin, Snail expression was decreased. The invasive abilities of Panc-1 cells in hpoxia environment was repressed greatly accordingly.Conclusion Hypoxia can activate the HIF-1αexpression which can induce the Snail transcriptional factor and trigger epithelial to mesenchymal transition.更多还原