【作者】 李云峰；

【导师】 林晨；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2008， 博士

【摘要】 恶性肿瘤严重威胁人类健康,亟需发展多种治疗方法改善肿瘤的预后。Gadd45a,在生长抑制和DNA损伤中具有重要作用,参与细胞生长的负性调控,瞬时转染Gadd45家族成员(Gadd45a,Gadd45g)可诱导肿瘤细胞的凋亡产生。本研究以腺病毒为载体,将Gadd45a导入胰腺癌细胞分析Gadd45a对胰腺癌细胞产生的生物学影响,分析Gadd45a对不同作用机制化疗药物的增效作用以及细胞p53状态对Gadd45a表达的影响;验证了Ad-G45a在裸鼠PANC1移植瘤上的抑制作用。结果:成功构建了表达Gadd45a的重组腺病毒Ad-G45a,经感染在四种人胰腺癌细胞(MIA PaCa-2,P3,PANC1,SW1990)中均可高效率表达。经测序分析未发现四种胰腺癌细胞的Gadd45a的cDNA有突变;在四种胰腺癌细胞系中PANC1的Gadd45a本底表达最高,但与细胞的恶性度没有明显相关性。以PANC1为研究对象,将Ad-G45a导入细胞后,细胞生长、侵袭和转移等恶性表型均受到明显抑制;Ad-G45a诱导细胞G2/M期阻滞具有明显的时效性和量效性;通过DAPI染色,annexinV,Westernblot等方法证实Gadd45a导入PANC1后通过激活Caspase9,诱导细胞凋亡。在PANC1细胞中证实Ad-G45a与化疗药物(cDDP,VP-16,5FU)合用有协同作用,以cDDP协同效应最为显著。外源Gadd45a的导入可提高野生型p53的表达水平,并诱导p53依赖的内源Gadd45a表达;VP-16在野生型p53的存在下可增强Ad-G45a诱导的内源Gadd45a表达。裸鼠PANC1移植瘤实验结果表明Ad-G45a具有明显抑瘤作用,抑瘤率达74.8%。更多还原

【Abstract】 The extremely poor prognosis of patients with pancreatic ductal adenocarcinoma(PDAC) indicates the need for novel therapeutic approaches. The growth arrest and DNA damage-inducible(Gadd) gene Gadd45a is a member of a group of genes that are induced by DNA damaging agents and growth arrest signals.In this report,we have analyzed the biological activity of Gadd45a upon pancreatic ductal adenocarcinoma cancer-derived cell lines.We report that Gadd45a is variously expressed in cell lines derived from pancreatic ductal adenocarcinoma cancer and adenoviral-mediated expression of Gadd45a (Ad-G45a) in these cells results in apoptosis via caspase activation and cell-cycle arrest in the G2/M phase.Furthermore,we assessed the efficacy of a combined treatment with Ad-G45a and anticancer drugs(Etoposide,cisplatin, 5-fluorouracil,respectively) for PANC1 cell line and found that Gadd45a significantly increased the chemosensitivity of PANC1 to chemotherapeutic agents,which may be resulted from abundant apoptosis induction and cell cycle arrest.By combinational treatment of Ad-G45a infection and chemotherapeutics, Gadd45a expression was elevated to a higher extent in cancer cells with wild-type p53 than in that with knocked-out p53,indicating a higher chemosensitivity to cancer chemotherapy.In PANC1 xenograft models, Ad-G45a significantly inhibited the growth of tumor(74.8%).Collectively,our results suggest that Gadd45a may paly nagative roles in cancer cell growth and may be a promising molecule used for the cancer gene therapy in combination with chemotherapeutic agents.更多还原