【作者】 陈芳；

【导师】 陈杰；

【作者基本信息】 中国协和医科大学 ， 病理学及病理生理学， 2005， 博士

【摘要】 胰腺癌是一种高度恶性的肿瘤，其发生发展与多种基因异常有关，如p53、K-ras、p16^INK4A、DPC4／SMAD4、BRCA2等。由于胰腺癌手术切除率低，放、化疗的效果差，探讨新的有效的治疗方法已成为胰腺癌研究的重要方向。我们采用重组腺病毒载体将外源性ARF／INK4a基因导入人胰腺癌细胞系中进行实验性基因治疗。 采用Western blot方法检测我实验室现有7株胰腺癌细胞系的p14^ARF和p16^INK4a的表达，发现其中PC-4、PC-7、Panc-1和Maia-Paca2的p14^ARF和p16^INK4a蛋白表达缺失，我们选取PC-7、Panc-1和Maia-Paca2进行转基因实验。 1．利用Hela细胞的mRNA，经RT-PCR得到野生型p14^ARF和p16^INK4a的cDNA，利用AdEasy重组腺病毒系统，构建复制缺陷型的重组腺病毒质粒pAdp14和pAdp16，经腺病毒包装细胞293细胞的包装、扩增后，得到复制缺陷型腺病毒Adp14和Adp16，分别感染PC-7、Panc-1和Maia-Paca2细胞，经Western Blot和RT-PCR证实各株细胞中有外源性的p14^ARF和p16^INK4a表达。 2．将Adp14分别感染PC-7、Panc-1和Maia-Paca2细胞，发现有野生型p53的PC-7细胞和p53突变的Maia-Paca2细胞生长速率减慢，G2期细胞比例增加，软琼脂克隆形成率降低，Western blot显示RB蛋白有不同程度去磷酸化，PC-7细胞p53表达上调；而p53突变的Panc-1细胞则未发生上述改变。经TUNEL法和流式细胞术未发现凋亡细胞比例明显上升。提示p14^ARF可通过依赖及不依赖p53的途径抑制胰腺癌细胞生长。 3．将Adp16分别感染PC-7、Panc-1和Maia-Paca2细胞，发现这三株细胞的生长速度均减低，出现G1期或G2期细胞比例增加，细胞出现衰老表型，Western blot显示RB蛋白磷酸化程度降低，E2F1表达上调，Panc-1和Maia-Paca2细胞软琼脂克隆形成率降低，经TUNEL法和流式细胞术未发现凋亡细胞比例明显上升。说明p16^INK4a通过RB途径抑制胰腺癌细胞生长。 4．在有野生型p53的PC-7细胞中可见Adp14和Adp16细胞生长抑制效果相似，联合使用这两种腺病毒与单独使用某一种病毒相比未见明显优势，Western blot显示与Adp16组相似的蛋白水平变化，TUNEL法和流式细胞术未发现凋亡细胞比例明显上升。更多还原

【Abstract】 The incidence of pancreatic cancer is increasing, so far neither an early diagnosis nor a therapeutic strategy for advanced lesions has been developed yet. It is an urgent mission for both the clinicians and the scientists who are challenging pancreatic cancer to find a breakthrough using new technologies. Recent progresses indicated that cancers are a group of diseases with accumulation of genetic alternations of oncogens and tumor suppressor genes. In pancreatic carcinoma, p53、K-ras、 p16INK4a、DPC4/SMAD4、BRCA2 have been considered as molecules that play key roles in tumourigenesis. In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we studied the effects of gene therapy by adenovirus-mediated transfer of p14ARF and p16INK4a into human pancreatic cancer cells.Western blot was used to determine the expression of p14ARF and p16INK4a in seven pancreatic carcinoma cell lines. Four cell lines were negative for both p14ARF and p16INK4a protein, and three of them (PC-7, Panc-1 and Maia-paca2) were used in our study of gene transfer.1. By RT-PCR technique, the wild-type cDNAs of p14ARF and p16INK4a were cloned and amplified from Hela cell. The recombinant replication-deficient adenoviral vectors Adp14 and Adp14, which contain p14ARF and p16INK4a cDNA respectively, were constructed by AdEasy System and amplified in 293 packaging cells. The pancreatic cancer cells transfected with Adp14 and Adpl6 showed expression of p14ARF and p16INK4a, respectively.2. PC-7 (p53+) and Maia-Paca2 (p53 mutant) transfected with Adp14 showed significant inhibition of cell growth and soft-agar colony formation as compared with control, G2 arrest by flow cytometry and dephosphorylation of RB protein by Western blot, but no obvious increase in apoptotic cells as detected by flow cytometry and TUNEL methods. In addition, PC-7 has elevated expression of p53 after treatment with Adp14. Panc-1, which also had mutant p53, did not show the same effects when transfected by Adp14. These results suggest that p14ARF can inhibit the growth of pancreatic cancer cells through p53 dependent as well as p53 independent pathways.更多还原