摘要
目的:研究利用低剂量腺病毒提高重组腺相关病毒2型(recombined adeno-associated virus,rAAV2)在耐药与非耐药肿瘤细胞中基因表达水平的新方法,并初步探讨其可能的作用机制。方法:rAAV2-GFP单独或联合复制缺陷型腺病毒(Ad5-RFP)或条件复制型腺病毒(Ad5-TERT-RFP)感染人非小细胞肺癌细胞系(NCI-H446)、人肺腺癌细胞系(A549)、人胃癌细胞系(SGC7901)、人口腔黏膜上皮癌细胞系(KB)和人口腔黏膜上皮癌耐长春新碱细胞系(KB/VCR)。荧光显微镜观察和流式细胞仪分析肿瘤细胞感染后GFP的表达;Western blotting检测感染后肿瘤细胞GFP蛋白表达及ERK和AKT磷酸化水平;Real-time PCR检测肿瘤细胞内GFP的mRNA表达量、DNA拷贝数,以及细胞表面受体HSPG、α_v integrin和FGFR-1的mRNA表达。结果:流式细胞结果显示,rAAV2-GFP联合Ad5-RFP或Ad-TERT-RFP感染肿瘤细胞24h后,GFP阳性细胞率和GFP平均荧光亮度分别提高了约0.3～3倍和4～8倍。Western blotting证实联合应用Ad5-RFP感染肿瘤细胞后24h,GFP蛋白表达增加约4～6倍,肿瘤细胞内ERK和AKT磷酸化水平升高。联合感染后细胞内GFP的mRNA表达量提高了3.83～7.33倍,DNA拷贝数未见明显改变,HSPG、α_v integrin和FGFR-1的mRNA表达轻微提高。结论:低剂量腺病毒显著提高rAAV2在耐药与非耐药肿瘤细胞中的基因表达水平,可能与激活信号传导通路、增加细胞内转录有关。
Objective： To investigate the enhancing effect of low dose adenovirus on rAAV2 expression in drug-sensitive and drug-resistant tumor cells and to explore the related mechanism. Methods：Human small cell lung cancer NCI- H446 cells, human lung adenocarcinoma A549 cells, human gastric cancer SGC7901 cells, human oral epithelial cancer KB cells, and VCR-resistant KB cells （KB/VCR） were transfected with rAAV2-GFP alone or combined with AdS-RFP or with AdS-TERT-RFP. GFP expression in the infected tumor cells was observed and analyzed by fluorescence microscope and fluorescence activated cell sorting （FACS）. GFP protein and phosphorylation level of ERK and AKT in the infected tumor cells were detected by Western blotting. Quantitative analysis of DNA copies of GFP and mRNA expression of GFP, HSPG, αv integrin , FGFR-1 in the infected tumor cells were performed by Real-time PCR. Results. The results of FACS demonstrated that the mean intensity of GFP fluorescence and the GFP positive rate after infection with rAAV2-GFP combined with Ad5-RFP or Ad5-TERT-RFP increased by 0.3-3 and 4-8 folds, respectively. GFP expression in the tumor cells with combined infection showed 4-6 folds increase and the phosphorylation level of ERK and AKT also increased. GFP mRNA expression in the combined infection tumor cells increased by 3.83-7.33 folds ; there was no differences in the GFP DNA copies between rAAV2-GFP group and rAAV2-GFP plus Ad5-RFP group. Cellular receptors HSPG, αv integrin and FGFR-1 mRNA expression was slightly increased in rAAV2-GFP plus Ad5-RFP. Conclusion ： Lower d^se of recombinant adenovirus can obviously enhance the expression of rAAV2 in drug-sensitive and drug-resistant tumor cells, which might be related to the activation of signal transduction pathway and increase of intracellular transcription.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2007年第5期405-410,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金杰出青年资助项目(No.30325043)
国家重点基础研究发展(973)计划资助项目(No.2004CB518804)
上海市卫生局医学领军人才基金
上海市科委攻关计划(No.064119539)
Supported by the National Natural Science Foundation of China(No.30325043)
The Major State Basic Research Development Program(973)of China(No.2004CB518804)
the Shanghai Outstanding Field Leaders Program
Key Science and Technology Research Project of Shanghai(No.064119539)
关键词
重组腺相关病毒
复制缺陷型腺病毒
条件复制型腺病毒
肿瘤细胞
基因表达
recombined adeno-associated virus
replication-defective adenovirus
replication-conditional adenovirus
tumor cells
gene expression
