【作者】 张建军；

【导师】 吴河水；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的研究Toll样受体4(TLR4)和低氧诱导因子-1α(HIF-1α)在胰腺癌中的表达及其临床意义。方法采用实时荧光定量PCR检测30例新鲜胰腺癌及相应癌旁组织标本中TLR4和HIF-1αmRNA水平,应用免疫组化检测TLR4、NF-κB p65和HIF-1α在65例胰腺癌及相应38例癌旁组织中的表达,分析其与临床病理特征间的关系以及TLR4和HIF-1α表达的相关性。应用Kaplan-Meier生存分析研究TLR4、HIF-1α蛋白表达对患者生存时间的影响。结果实时荧光定量PCR结果表明胰腺癌组织中的TLR4、HIF-1αmRNA水平分别为0.81±0.10、0.87±0.11,显著高于癌旁组织(0.70±0.16、0.68±0.13,p<0.05)；免疫组化结果显示TLR4、NF-κB p65和HIF-1α蛋白在胰腺癌组织中的表达率分别为69.20%、66.15%和70.80%,显著高于癌旁组织(39.50%、31.58%和36.8%,p<0.05),TLR4和HIF-1α的表达与患者性别、年龄、肿瘤部位及分化程度无关,而与肿瘤大小、淋巴结转移、血管侵犯及临床TNM分期相关。TLR4和HIF-1α表达呈正相关。TLR4或HIF-1α阴性组患者的生存时间显著长于TLR4或HIF-1α阳性组。结论TLR4、NF-κB p65和HIF-1α在胰腺癌中高表达,TLR4可能通过NF-κBp65途径参与了HIF-1α的表达调控,共同促进了胰腺癌的恶性进展。目的构建针对人TLR4基因的shRNA真核表达质粒,并筛选出对胰腺癌细胞系PANC-1基因沉默效果最明显的shRNA质粒表达载体,转染胰腺癌细胞并筛选出稳定转染的克隆细胞株。方法针对TLR4基因的mRNA序列设计,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切,测序鉴定,脂质体法转染至胰腺癌PANC-1细胞,48h后实时荧光定量PCR检测TLR4 mRNA被抑制情况。将抑制效率最高的shRNA质粒转染PANC-1细胞,G418抗性筛选和有限稀释单克隆形成法挑选培育稳定转染TLR4 shRNA的单克隆细胞系,实时定量PCR和流式细胞技术检测单克隆细胞系中TLR4 shRNA转染效率和基因沉默效率。结果经测序证实,成功构建TLR4 shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致。重组质粒转染PANC-1细胞48h后,转染效率为46.72%±5.06%, TLR4 mRNA水平明显下调；其中以TLR4 RNAi-3#重组质粒的沉默效应最强(0.019±0.006 vs 0.061±0.018,P=0.000)。将TLR4 RNAi-3#转染至PANC-1细胞,G418抗性筛选和有限稀释单克隆形成法形成稳转单克隆细胞系,与转染48h比较,转染效率显著提高(82.79%±8.16% vs 46.72%±5.06%, P=0.001), TLR4 mRNA显著被抑制(0.010±0.002 vs 0.019±0.006,P=0.01),稳转细胞TLR4蛋白表达率显著低于PANC-1细胞或转染阴性质粒细胞(0.54%±0.32%vs 87.42%±5%；0.54%±0.32%vs 82.9%±5%,P=0.000)。结论成功构建了携带以TLR4为靶向的shRNA的重组质粒,其对胰腺癌PANC-1细胞TLR4的表达具有显著抑制效应,TLR4 shRNA稳定转染细胞的转染效率以及基因沉默效率显著高于转染48h。该实验为进一步研究TLR4的功能提供了实验基础。目的研究胰腺癌细胞系PANC-1中TLR4信号通路对HIF-1αmRNA和蛋白表达的影响及调控机制。方法分别将PANC-1细胞、转染阴性对照质粒的PANC-1 RNAi Ctro细胞以及转染shRNA TLR4表达质粒的PANC-1 RNAi TLR4细胞用低氧模拟剂氯化钻(CoCl2,100μmol/L)、不同浓度的TLR4配体脂多糖(LPS, 0μg/ml, 1μg/ml,10μg/ml,)和NF-K B特异性抑制剂PDTC (25μM)进行不同时间(6h,12h,24h,48h)的干预,实时荧光定量PCR检测细胞中HIF-1αmRNA水平,免疫印迹检测HIF-1α蛋白表达情况。分别用LPS (1μg/ml, 10μg/ml)作用PANC-1细胞及PANC-1 RNAi TLR4细胞30min,电泳迁移率实验(EMSA)分析细胞中的NF-κB活性。结果胰腺癌细胞中LPS以时间和剂量依赖的方式上调了HIF-1αmRNA及蛋白的表达。NF-κB抑制剂PDTC和TLR4基因沉默均显著抑制了LPS诱导的HIF-1α表达,LPS诱导了PANC-1细胞中NF-KB的活化。结论TLR4信号通路通过NF-κB途径参与了HIF-1α的表达上调。目的研究TLR4信号通路对胰腺癌细胞粘附和侵袭能力的影响极其机制方法分别对PANC-1细胞、转染阴性对照质粒的PANC-1 RNAi Ctro细胞以及转染shRNA TLR4表达质粒的PANC-1 RNAi TLR4细胞用不同浓度的LPS和NF-κB特异性抑制剂PDTC进行不同时间的干预,粘附实验检测细胞和基质胶MaxGel的粘附率,Transwell小室侵袭实验检测干预后细胞的侵袭力,流式细胞技术检查癌细胞中整合素β1 (Integrinβ1)的表达,ELISA检测细胞培养上清夜中基质金属蛋白酶-9(MMP-9)的浓度,细胞免疫荧光实验观察LPS作用后PANC-1细胞中NF-κB核转位情况。结果LPS以时间和剂量依赖的方式促进了PANC-1细胞与基质胶的粘附,并增强了细胞侵袭力。NF-κB抑制剂PDTC和TLR4基因沉默抑制了细胞的粘附率及侵袭力。LPS上调了PANC-1细胞中Integrinβ1蛋白的表达,促进细胞分泌MMP-9。NF-κB抑制剂PDTC抑制了LPS对PANC-1细胞Integrinβ1和MMP-9表达的诱导作用。LPS未能引起PANC-1 RNAi TLR4细胞中Integrinβ1和MMP-9表达上调。LPS促进了PANC-1细胞NF-κB p65核转位,但对PANC-1 RNAi TLR4细胞未见LPS对NF-κB p65核转位的诱导。结论TLR4信号通路上调了胰腺癌细胞中整合素β1和基质金属蛋白酶-9的表达,从而促进了癌细胞与细胞外基质的粘附,增加了癌细胞的侵袭力。更多还原

【Abstract】 AIM:To investigate the expression of Toll-like receptor (TLR) 4, nuclear factor-kappaB(NF-κB) p65 and hypoxia-inducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma.METHODS:TLR4 mRNA and HIF-1αmRNA were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues, and expression of TLR4, NF-κB p65 and HIF-1αprotein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues. The relationship between TLR4 or HIF-1αand pathologic features, association between TLR4 and HIF-1α, were also analyzed. Kaplan-Meier Method was used to assess the impact of TLR4 and HIF-1αexpression on survival of patients.RESULTS:The relative quantification of TLR4 mRNA and HIF-1αmRNA in tumor tissues were 0.81±0.10 and 0.87±0.11 respectively, which were significantly higher than that in adjacent tissues (0.70±0.16 and 0.68±0.13 respectively, P<0.05). The protein expression of TLR4, NF-κB p65 and HIF-1αin tumor tissues were 69.20%,66.15% and 70.80% respectively, significantly higher than that in adjacent normal tissues(39.50%, 31.58% and 36.80%, respectively, P< 0.05). There were no significant correlation between TLR4 or HIF-1αexpression and the age, gender, tumor location, the degree of tumor differentiation in patients (P>0.05). However, there were significant correlation between the expression of TLR4 or HIF-1αand tumor size, lymphnode metastasis, venous invasion and clinical staging(P<0.05). The expression of TLR4 and HIF-1αhad a significant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4, NF-κB p65 and HIF-1αare overexpressed in pancreatic adenocarcinoma, TLR4 may be partly involved in up-regulating HIF-1α, and both synergyly accelerate progresis of pancreatic adenocarcinoma.AIM:To investigate the effects of eukaryotic plasmic expression vector mediated short hairpin RNA interference targeting TLR4 gene on pancreatic adenocarcinoma cells.METHODS:We constructed three short hairpin RNA (shRNA) interference expression plasmid vectors targeting the TLR4 gene and a control vector containing random DNA fragment.The recombinant plasmids were amplified in E.coli. DH5 a was identified by restriction digestion, PCR and sequencing. The vevtors were tansfected into PANC-1 cells. TLR4 mRNA was detected by real-time PCR, the most effective vector TLR4 RNAi-3# was transfected into PANC-1 and stably transfected clones was obtained by Geneticin (G418) and limiting dilution analysis. The transfection efficiency of plasmids were detected through evaluation of GFP by flow cytometry analysis, and the silencing effect of TLR4 was assayed with real-time quantitative PCR and Flow cytometry analysis.RESULTS:The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. At 48 h after transfection, the transfection efficiency of plasmids was 46.72%±5.06%, and TLR4 mRNA was significantly down-regulated, especially in cells transfected with TLR4 RNAi-3# vector (0.019±0.006 vs 0.061±0.018, P=0.000). The transfection efficiency of TLR4 RNAi-3# vector in stably transfected clones was significantly higher than that of transient transfection (82.79%±8.16%vs 46.72%±5.06%, P=0.001). TLR4 mRNA was absolutely silenced (0.010±0.002 vs 0.019±0.006, P=0.01). The expression of TLR4 protein on stably transfected clones was significantly lowed than that on PANC-1 cells or cells transfected with control plasmid(0.54%±0.32% vs 87.42%±5%; 0.54%±0.32% vs 82.9%±5%, P=0.000).CONCLUSION:Plasmid vector expressing shRNA against TLR4 has been successfully constructed and it can down-regulated TLR4 on pancreatic adenocarcinoma effctively.AIM:To explore the role of the crosstalk of TLR4 and HIF-1αin pancreatic ductal adenocarcinoma and the possible mechanism.METHODS:The expression of TLR4 and HIF-1αprotein were detected by immunohistochemistry in pancreatic ductal adenocarcinoma and its adjacent tissues. Then the expression of HIF-1αwas observed in the human pancreatic cancer cell line PANC-1 by real time PCR and Western blotting after treated with cobalt chloride (CoC12, a hypoxia mimicking agent), LPS and PDTC(NF-kappa B inhibitor) respectively. RNA interference expression vector for TLR4 was transfected into PANC-1 cells, and the stably transfected clones were treated with CoC12 and LPS, then the expression of HIF-la was detected by real time PCR and Western blotting, and the NF-kappa B p65 nuclear translocation in was analyzed using immunofluorescence staining.RESULTS:The protein expression of TLR4 and HIF-la in tumor tissues were 69.20% and 70.80% respectively, significantly higher than that in adjacent normal tissues respectively (69.2% v.s 39.5%, P=0.003;70.8% v.s 36.8%, P=0.001). HIF-1αexpression was positive correlation with TLR4 (rp=0.451, p<0.05). LPS and CoC12 significantly increase HIF-la mRNA and protein in PANC-1 cells compared with CoC12 alone, both silencing of TLR4 and a specific NF-κB inhibitor, PDTC, suppressed the increase of HIF-la induced by LPS. Nuclear translocation of NF-κB p65 was induced by LPS in PANC-1 cells.CONCLUSION:TLR4 and HIF-1αare overexpressed in pancreatic adenocarcinoma TLR4 signaling regulates HIF-la mRNA and protein expression via NF-κB activation in pancreatic tumor microenviroment.AIM:To investigate the role of TLR4 signal pathway in adhesion and invasion of human pancreatic cancer cells to cell-extracellular matrix.METHODS:PANC-1 cells, PANC-1 RNAi Ctro cells and PANC-1 RNAi TLR4 cells were stimulated with LPS respectively, in vitro tumour cell ECM adhesion and ECM invasion were analysed by ECM adhesion assay and ECM invasion chambers. The expression of Integrinβ1 and their alteration by LPS were examined by flow-cytometric analysis, and the level of MMP-9 in cellular supernatant was detected by enzyme-linked immunosorbent assay. Nuclear translocation of the NF-κB p65 induced by LPS was observed by immunofluorescence staining.RESULTS:PANC-1 cancer cells showed preferential tumour cell ECM adhesion and ECM invasion compared with PANC-1 RNAi TLR4 cells, and this was enhanced by LPS. These effects were abolished by shTLR4 and NF-κB inhibition. LPS upregulates Integrinβ1 and MMP-9 in a dose-dependent and time-dependent manner, and the expression of Integrinβ1 and MMP-9 was decreased by NF-kB inhibition and shRNA. Nuclear translocation of the NF-κB p65 is induced by LPS in PANC-1.CONCLUSION:In pancreatic cancer, LPS promotes tumour cell ECM adhesion and invasion through activation of Integrinβ1 and MMP-9 in a TLR-4- and NF-κB-dependent manner.更多还原