【作者】 宋建元；

【导师】 曹建平；

【作者基本信息】 苏州大学 ， 放射医学， 2017， 博士

【摘要】 目的胰腺癌是常见恶性肿瘤。放疗是胰腺癌治疗的重要手段,因而增加胰腺癌细胞的放射敏感性具有重要意义。非诺贝特（fenofibrate）是PPARα的特异性激动剂。本研究拟探讨PPARα在胰腺癌中的表达,PPARα激动剂非诺贝特联合X射线对人胰腺癌PANC1细胞、Patu8988细胞生长和放射敏感性的影响及机制研究。方法通过免疫组化法检测80例胰腺癌及癌旁组织中PPARα的差异表达,并观察其与胰腺癌患者年龄、性别、TNM分期的关系。通过MTT检测非诺贝特联合X射线对人胰腺癌PANC1细胞、Patu8988细胞生长的影响;平板克隆形成实验检测检测非诺贝特联合X射线对人胰腺癌PANC1细胞、Patu8988细胞克隆形成的影响;通过划痕实验检测非诺贝特和X射线联合作用对PANC1细胞和Patu8988细胞迁移的影响;通过Transwell细胞侵袭实验检测非诺贝特和X射线联合作用对PANC1细胞和Patu8988细胞侵袭能力的影响;通过裸鼠皮下建立人胰腺癌PANC1移植瘤模型,检测非诺贝特和X射线联合作用对裸鼠移植瘤生长的影响;通过免疫荧光法检测非诺贝特对PANC1细胞PPARα表达的影响;通过表达谱芯片筛选非诺贝特联合X射线照射后PANC1细胞差异表达的mRNA;通过MTT检测ZnCl₂、TPEN对人胰腺癌细胞株PANC1生长的影响;通过锌离子探针方法观察非诺贝特和X射线联合作用对PANC1细胞锌离子的影响;通过Western Blot检测非诺贝特对PANC1细胞ZIP7表达的影响;通过免疫组织化学方法分析80例胰腺癌组织和胰腺癌旁组织中ZIP7表达水平。结果PPARα在胰腺癌组织表达水平高于癌旁组织（P<0.01）,胰腺癌组织中PPARα蛋白表达水平与TNM分期有关（P<0.01）,与年龄、性别无关;非诺贝特能够增强X射线对人胰腺癌PANC1细胞和Patu8988细胞生长、克隆形成、迁移、侵袭以及裸鼠PANC1移植瘤生长的抑制;非诺贝特能够促进PPARα在PANC1细胞核表达;非诺贝特联合X射线照射后PANC1细胞差异表达的mRNA包括TAOK2,JAK3,SLC39A7（ZIP7）和TRPV1等2669个;表达的基因影响多个通路。ZnCl₂促进PANC1细胞生长,金属螯合剂TPEN能够抑制PANC1细胞生长;非诺贝特和X射线联合作用能够促进PANC1细胞锌离子在细胞内聚集减少;非诺贝特抑制PANC1细胞中ZIP7表达;ZIP7在胰腺癌组织表达水平高于癌旁组织。结论PPARα胰腺癌组织中高表达,并与临床病理特征和生存期相关。PPARα激动剂非诺贝特能够增强胰腺癌细胞放射敏感性;非诺贝特能直接或间接影响胰腺癌细胞中多个基因表达。非诺贝特可抑制ZIP7表达,降低胰腺癌细胞内锌离子浓度,并促使某些关键蛋白缺锌,从而引起细胞死亡增加,这可能是非诺贝特增加胰腺癌细胞放射敏感性途径之一。更多还原

【Abstract】 Objective: Radiotherapy plays a significant role in the management of pancreatic cancer.Enhancing the radiation sensitivity of pancreatic cells will be beneficial for the treatment of pancreatic cancer patients.Fenofibrate is a specific ligand for nuclear receptor peroxisome proliferator-activated receptor alpha(PPARα),which is involved in multiple pathophysiological processes.This study aims to investigate the expression level of PPARα in pancreatic cancer and the radiosensitivity effect of PPARα activation fenofibrate on human pancreatic cancer cells and its underlying mechanisms.Methods: 80 pancreatic cancer tissue samples were collected.The expression of PPARα in pancreatic cancer tissues were performed by immunohistochemistry.Pancreatic cancer cells PANC1 and Patu8988 were treated with fenofibrate and/or IR(radiation),and the cell viability was detected by MTT assay.Radiosensitivity of cells was determined using colony formation assay.The migration ability was detected by scratch migration assay.The invasion ability was detected by transwell assay.The nude mice were subcutaneous implantated with human pancreatic cancer PANC1 cells.Then,the nude mice were treated with fenofibrate and/or IR.The tumor growth was measured every three days.After PANC1 cells were treated with fenofibrate,immunofluorescence staining were performed to detect thedistribution of PPARα.mRNA microarray was used to explore the involved genes.The intracellular Zn2+ accumulation was detected by Zn2+ fluorescent probe.Results: The expression of PPARα was significantly higher in pancreatic cancer tissues,compared with the para-tissues tissues(P < 0.01).The expression of PPARα was correlated with the TNM stage(P < 0.01).PPARα agonist fenofibrate inhibited the proliferation of human pancreatic cell lines(PANC1 and Patu8988)in a dose-dependent manner.Pretreatment of 50 μM fenofibrate in PANC1 and Patu8988 cells potentiated the anticancer effect of radiation.In addition,pretreatment of fenofibrate combined with radiation significantly suppressed cell migration and invasion abilities in pancreatic cancer cell lines.Moreover,fenofibrate sensitized PANC1 xenografts to irradiation with an enhancement factor 2.10(P < 0.05).Microarray analysis revealed that fenofibrate plus radiation affected 2669 genes(Fold-change > 2.0),including TAOK2,JAK3,SLC39A7(ZIP7)and TRPV1.Pathway analysis demonstrated that fenofibrate affected multiple pathways,including cytokine-cytokine receptor interaction,RIG-I-like receptor signaling pathway and transcriptional misregulation in cancer.Taken together,these results suggest that fenofibrate enhances the radiosensitivity of human pancreatic cancer cells in vitro and in vivo.Specifically,we focused on ZIP7,which was decreased by fenofibrate.Knock-down of ZIP7 decreased Zn2+ accumulation in pancreatic cancer cells.Consistently,pretreatment of fenofibrate combined with radiation significantly suppressed zinc aggregation in PANC1 cell,which is likely to induce cell death due to lack of zinc.Finally,we found that the expression of ZIP7 was significantly increased in pancreatic cancer tissues,compared with the para-tissues tissues.Conclusions: The expression level of PPARα is increased in pancreatic cancer.PPARα agonist fenofibrate increases the radiosensitivity of human pancreatic cancer cells.Fenofibrate plus radiation affected multiple genes that implicated in multiple pathways.Fenofibrate can inhibit the expression of ZIP7,decreasing zinc aggregation in cytoplasm.更多还原