肿瘤
腫瘤
종류
TUMOR
2009年
7期
603-610
,共8页
刘新建%季迅达%田毓华%陈霞芳%谢匡成%吴继红%张圣海%徐萍%李川源%黄倩
劉新建%季迅達%田毓華%陳霞芳%謝劻成%吳繼紅%張聖海%徐萍%李川源%黃倩
류신건%계신체%전육화%진하방%사광성%오계홍%장골해%서평%리천원%황천
胃肿瘤%腺病毒感染%基因表达调控,肿瘤%缺氧诱导因子1%二氯化钴
胃腫瘤%腺病毒感染%基因錶達調控,腫瘤%缺氧誘導因子1%二氯化鈷
위종류%선병독감염%기인표체조공,종류%결양유도인자1%이록화고
Stomach neoplasms%Adenoviridae infections%Gene expression regulation,neoplastic%Hypoxia-inducible factor 1%CoCl2
目的:研究二氯化钴(CoCl2)对缺氧反应元件调控E1AE1B表达的条件复制型腺病毒载体(Ad-5HRE-E1AE1B-RFP)和缺乏缺氧反应元件的复制缺陷型腺病毒载体(Ad-EGFP)在肿瘤细胞内表达和复制的影响. 方法:肿瘤细胞经不同浓度的CoCl2 处理后,Western 印迹法检测缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的表达;倒置荧光显微镜、FCM法及空斑形成实验等观察经CoCl2处理后,感染条件复制型腺病毒和(或)复制缺陷型腺病毒的肿瘤细胞中外源基因的表达水平和病毒复制情况;小动物成像仪观察缺氧调控的条件复制型腺病毒在肿瘤内提高复制缺陷型腺病毒表达的效率. 结果:适当浓度的CoCl2 (0.4和0.08 μg/mL) 能使胃癌细胞株(SGC7901)中HIF-1α蛋白稳定表达并积聚,可较好地模拟缺氧状态;在0.4 μg/mL CoCl2作用下,Ad-5HRE-E1AE1B-RFP对肿瘤细胞的感染效率明显提高,表现为外源基因RFP阳性表达的百分率和荧光强度均明显提高,但空斑形成实验显示Ad-5HRE-E1AE1B-RFP并没有复制.0.4 μg/mL CoCl2也能提高其他非缺氧调控的复制缺陷型腺病毒如Ad-EGFP、Ad-Luc的感染效率和表达水平; Ad-5HRE-E1AE1B-RFP与复制缺陷型腺病毒Ad-Luc联合注射裸鼠肿瘤能显著提高Ad-Luc的表达.结论:CoCl2 能明显提高腺病毒的基因表达水平,其作用机制不仅与CoCl2诱导缺氧有关,也可能与影响基因转录有关.
目的:研究二氯化鈷(CoCl2)對缺氧反應元件調控E1AE1B錶達的條件複製型腺病毒載體(Ad-5HRE-E1AE1B-RFP)和缺乏缺氧反應元件的複製缺陷型腺病毒載體(Ad-EGFP)在腫瘤細胞內錶達和複製的影響. 方法:腫瘤細胞經不同濃度的CoCl2 處理後,Western 印跡法檢測缺氧誘導因子(hypoxia inducible factor-1α,HIF-1α)的錶達;倒置熒光顯微鏡、FCM法及空斑形成實驗等觀察經CoCl2處理後,感染條件複製型腺病毒和(或)複製缺陷型腺病毒的腫瘤細胞中外源基因的錶達水平和病毒複製情況;小動物成像儀觀察缺氧調控的條件複製型腺病毒在腫瘤內提高複製缺陷型腺病毒錶達的效率. 結果:適噹濃度的CoCl2 (0.4和0.08 μg/mL) 能使胃癌細胞株(SGC7901)中HIF-1α蛋白穩定錶達併積聚,可較好地模擬缺氧狀態;在0.4 μg/mL CoCl2作用下,Ad-5HRE-E1AE1B-RFP對腫瘤細胞的感染效率明顯提高,錶現為外源基因RFP暘性錶達的百分率和熒光彊度均明顯提高,但空斑形成實驗顯示Ad-5HRE-E1AE1B-RFP併沒有複製.0.4 μg/mL CoCl2也能提高其他非缺氧調控的複製缺陷型腺病毒如Ad-EGFP、Ad-Luc的感染效率和錶達水平; Ad-5HRE-E1AE1B-RFP與複製缺陷型腺病毒Ad-Luc聯閤註射裸鼠腫瘤能顯著提高Ad-Luc的錶達.結論:CoCl2 能明顯提高腺病毒的基因錶達水平,其作用機製不僅與CoCl2誘導缺氧有關,也可能與影響基因轉錄有關.
목적:연구이록화고(CoCl2)대결양반응원건조공E1AE1B표체적조건복제형선병독재체(Ad-5HRE-E1AE1B-RFP)화결핍결양반응원건적복제결함형선병독재체(Ad-EGFP)재종류세포내표체화복제적영향. 방법:종류세포경불동농도적CoCl2 처리후,Western 인적법검측결양유도인자(hypoxia inducible factor-1α,HIF-1α)적표체;도치형광현미경、FCM법급공반형성실험등관찰경CoCl2처리후,감염조건복제형선병독화(혹)복제결함형선병독적종류세포중외원기인적표체수평화병독복제정황;소동물성상의관찰결양조공적조건복제형선병독재종류내제고복제결함형선병독표체적효솔. 결과:괄당농도적CoCl2 (0.4화0.08 μg/mL) 능사위암세포주(SGC7901)중HIF-1α단백은정표체병적취,가교호지모의결양상태;재0.4 μg/mL CoCl2작용하,Ad-5HRE-E1AE1B-RFP대종류세포적감염효솔명현제고,표현위외원기인RFP양성표체적백분솔화형광강도균명현제고,단공반형성실험현시Ad-5HRE-E1AE1B-RFP병몰유복제.0.4 μg/mL CoCl2야능제고기타비결양조공적복제결함형선병독여Ad-EGFP、Ad-Luc적감염효솔화표체수평; Ad-5HRE-E1AE1B-RFP여복제결함형선병독Ad-Luc연합주사라서종류능현저제고Ad-Luc적표체.결론:CoCl2 능명현제고선병독적기인표체수평,기작용궤제불부여CoCl2유도결양유관,야가능여영향기인전록유관.
Objective: To investigate the effect of cobalt chloride (CoCl2) on transgene expression and viral particle titers in tumor cells infected by conditionally replicating adenovirus expression vector with hypoxia response elements(HRE)-regulated E1AE1B expression (Ad-5HRE-E1AE1B-RFP) and non-HRE regulated replication-deficient adenovirus expression vector (Ad-EGFP, Ad-Luc) in vitro and in vivo. Methods: Ad-5HRE-E1AE1B-RFP had five duplicated HRE and mini CMV acted as a promoter to drive E1AE1B expression and constitutive expression of RFP as reporter. The hypoxia model was optimized by exposing tumor cells to different concentrations of CoCl2. The hypoxia-inducible factor 1 alpha (HIF-1α) protein expression was determined by Western blotting. Under the optimized hypoxia model, the positive expression of exogenous gene and virus replication of Ad-5HRE-E1AE1B-RFP or Ad-EGFP-infected tumor cells were examined by conversed microscopic observation, FACS analysis and plaques formation test. Furthermore, transgene expression induced by combined application of hypoxia-inducible replicative adenovirus and replication deficient adenovirus (Ad-Luc) was also evaluated by examining the lucifererse activity in xenografted tumor models in nude mice by micro PET. Results: Western blotting results showed that CoCl2 at 0.4 and 0.08 μg/mL could stabilize and acumulate HIF-1α protein in gastric cancer SGC7901 cells, which could better mimic hypoxia condition. The microscopic observation and FACS analysis showed that CoCl2 at 0.4 μg/mL could remarkably increase the transduction efficacy of Ad-5HRE-E1AE1B-RFP, which was verified by significant increase in the percentage of positive expression of exogenous gene RFP and fluorescence intensity. But plaques formation test showed that Ad-5HRE-E1AE1B-RFP had no replication. CoCl2 0.4 μg/mL augmented the tranduction efficacy and expression levels of non-HRE regulated replication deficient adenovirus Ad-EGFP and Ad-Luc. Combined intratumoral injection of Ad-5HRE-E1AE1B-RFP and Ad-Luc significantly increased the expression of Ad-Luc in nude mice.Conclusion: CoCl2 markedly enhances transgene expression of recombinant adenovirus. However, the underlying mechanism is not only related to the CoCl2-induced hypoxia, but also probably related to regulation of gene transcription.
