摘要
目的构建p53正向凋亡调节因子重组腺病毒（Ad-PUMA）载体，探讨Ad—PUMA对人体内外胰腺癌的治疗作用。方法利用Ad—Easy系统在大肠杆菌同源重组，构建Ad—PUMA腺病毒载体，在293细胞内成功包装并鉴定后，以Ad-PUMA转染人转移胰腺癌细胞株AsPC-1。用MTT法检测转染前后存活细胞，观察Ad．PUMA的体外抑瘤作用；用Western blot方法鉴定转染前后AsPC-1细胞内PUMA蛋白表达。通过裸鼠皮下胰腺癌移植瘤模型观察Ad-PUMA的体内抑瘤效果；Western blot方法鉴定Ad—PUMA转染后肿瘤组织内PUMA蛋白表达，TUNEL（terminal deoxynucleotidyl transferase biotin—dUTP nick end labeling）法检测肿瘤组织细胞凋亡。结果在体外，随着Ad—PUMA感染剂量增加，细胞内PUMA蛋白表达逐渐增加，细胞存活率逐渐下降；在体内，Ad—PUMA可显著抑制裸鼠皮下肿瘤的生长，其抑瘤率为44．2％，瘤体组织PUMA表达水平及凋亡指数明显升高。结论PUMA抑制体内外胰腺癌细胞增殖，可能是-种潜在的肿瘤生物治疗手段。
Objective To construct the recombinant adenovirus containing p53 up-regulated mod- ulator of apoptosis （Ad-PUMA） and investigate its growth inhibition effect on pancreatic cancer cells in vitro and in vivo. Methods Ad-Easy system was used to construct Ad-PUMA by recombination in E. coli. The virus was packaged in 293 cells and subsequently identified valid. The AsPC-1 cells were infected with Ad- PUMA. Before and after Ad-PUMA infection, the expression of PUMA protein was investigated by Western blot, the inhibition rate of AsPC-1 cells was examined by MTT assay. The in vivo tumor suppressive effect was detected in nude mice with human AsPC-1 xenograft. PUMA protein and the apoptosis of AsPC-1 xeno- graft were detected by Western blot and TUNEL（ terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling） method. Results In vitro, the expression of PUMA protein was increased with titer of Ad-PUMA, the proliferation of AsPC-1 cells were suppressed, significantly, and the effect was in a viral dose-dependent manner. In vivo, the growth in nude mice of AsPC-1 infected with Ad-PUMA was significantly inhibited with an inhibition rate of 44.2%. The expression of PUMA was significantly up-regulated, and the apoptosis in- dex was significantly increased in tumor after Ad-PUMA infection as determined by Western blot and TUNEL. Conclusion The expression of PUMA can inhibit the proliferation of pancreatic cancer in vitro and in vivo, and may be used as a potential tool for cancer therapy
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第1期65-70,共6页
Chinese Journal of Microbiology and Immunology
基金
基金项目：卫生部科学研究资金项目（2004-002-1）
关键词
腺病毒载体
p53正向凋亡调节因子
基因治疗
细胞凋亡
胰腺肿瘤
Adenovirus vector
p53 up-regulated modulator of apoptosis（PUMA）
Gene therapy
Apoptosis
Pancreatic carcinoma
