【作者】 何小平；

【导师】 李兆申； 许国铭； 屠振兴； 钱其军；

【作者基本信息】 第二军医大学 ， 内科学， 2006， 博士

【摘要】 本课题旨在将抗血管生成治疗与基因治疗相结合,探讨胰腺癌治疗新策略。首先利用逆转录多聚酶链式反应(RT-PCR)从人胎盘组织扩增出新的血管生成抑制剂基因canstatin,插入质粒pET-22b(+)构建原核表达载体pET/canstatin,转化大肠杆菌,在IPTG诱导下表达出重组人Canstatin蛋白,体内实验证实其能通过抑制血管生成有效抑制胰腺癌生长。继而,将canstatin基因插入载体pCA13和pXC7C,构建出穿梭载体pCA13-Cans和pXC7C-Cans,再分别与pBGHE3共转染293细胞,获得重组非增殖型腺病毒Ad5-Cans和增殖型腺病毒(溶瘤病毒)He-Cans。体内外实验证实,溶瘤病毒He-Cans能有效抑制胰腺癌生长,疗效强于Ad5-Cans和Canstatin蛋白,且副作用少,具有良好临床应用前景。其作用机制主要是:①表达Canstatin蛋白,抑制肿瘤新生血管形成;②在肿瘤细胞中特异性增殖并裂解肿瘤细胞,从而发挥溶瘤作用。更多还原

【Abstract】 The purpose of the present study is to develop new treatment strategy for pancreatic cancer through combining anti-angiogenesis therapy with gene therapy. The canstatin gene cDNA was amplified by RT-PCR from the total RNA extracted from human placenta, and was inserted into plasmid pET-22b(+) to construct prokaryotic expression vector pET/canstatin, which was induced to express Canstatin protein by IPTG after transformed into E. coli BL21. Our findings demonstrate that Canstatin protein effectively retards the growth of pancreatic cancer through inhibiting angiogenesis in vivo. Then, canstatin gene was inserted into vector pCA13 and pXC7C to construct shutter vector pCA13-Cans and pXC7C-Cans, which were co-transfected with pBHGE3 separately into 293 cells to produce new recombinant replication incompetent and competent adenoviruses, named as Ad5-Cans and He-Cans respectively. The results of the in vitro and in vivo experiments show that adenovirus He-Cans efficiently inhibits the growth of pancreatic cancer, far more potent than adenovirus Ad5-Cans and Canstatin protein without remarkable adverse effects, showing a promising future in its clinical application. The main mechanisms of its anti-tumor effect include anti-angiogenesis effect by expressing Canstatin protein and oncolytic effect by replicating selectively in tumor cells.更多还原