CAJ | 학술논문

目的通过体外实验探讨人端粒酶反转录酶(hTERT)反义寡核苷酸(ASODN)能否增强胰腺癌细胞对吉西他滨(健择)的敏感性.方法采用实时荧光定量逆转录聚合酶链反应(RT-PCR)法检测hTERT mRNA表达情况;端粒重复序列扩增法(TRAP)、聚丙烯酰胺凝胶电泳和银染法检测端粒酶活性;四甲基偶氮唑盐(MTT)法检测细胞增殖能力;膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)和碘化丙啶(PI)双染色法检测早期凋亡.结果瞬时转染hTERT ASODN呈浓度依赖性下调细胞hTERT mRNA表达.0.2 μmol/L hTERT ASODN可显著下调端粒酶活性至0.47.健择在ASODN转染组中的IC50值为(0.8±0.2)μmol/L,而其在单纯脂质体转染对照组中为(7.3±0.9)μmol/L,差异具有统计学意义.ASODN可显著增加健择对肿瘤细胞的凋亡诱导作用,健择诱导的早期凋亡率在ASODN转染组中为60.28%,而其在脂质体对照组中为17.34%.结论 hTERTASODN可增强胰腺癌细胞对健择的敏感性,其机制可能与hTERT mRNA和端粒酶活性下调相关.
목적 통과체외실험탐토인단립매반전록매(hTERT)반의과핵감산(ASODN)능부증강이선암세포대길서타빈(건택)적민감성.방법 채용실시형광정량역전록취합매련반응(RT-PCR)법검측hTERT mRNA표체정황;단립중복서렬확증법(TRAP)、취병희선알응효전영화은염법검측단립매활성;사갑기우담서염(MTT)법검측세포증식능력;막련단백V-이류청산형광소(Annexin V-FITC)화전화병정(PI)쌍염색법검측조기조망.결과 순시전염hTERT ASODN정농도의뢰성하조세포hTERT mRNA표체.0.2 μmol/L hTERT ASODN가현저하조단립매활성지0.47.건택재ASODN전염조중적IC50치위(0.8±0.2)μmol/L,이기재단순지질체전염대조조중위(7.3±0.9)μmol/L,차이구유통계학의의.ASODN가현저증가건택대종류세포적조망유도작용,건택유도적조기조망솔재ASODN전염조중위60.28%,이기재지질체대조조중위17.34%.결론 hTERTASODN가증강이선암세포대건택적민감성,기궤제가능여hTERT mRNA화단립매활성하조상관.
Objective To evaluate whether antisense oligonucleotides (ASODN) targeting hTERT mRNA could sensitize human pancreatic cancer cell to gemcitabine in vitro. Methods hTERT mRNA expression was measured by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Telomerase activity was examined by TRAP, polyacrylamide gel electrophoresis and silver staining. The proliferation capacity and the apoptosis of cancer cells were evaluated by MTT assay and double staining with both Annexin V-FITC and PI. Results Transient transfection in human pancreatic cancer cell with hTERT ASODN diminished the abundance of hTERT mRNA in a concentration-dependent fashion. And 0.2 μmoL/L ASODN decreased the telomerase activity by 0.47-fold in cancer cell. The IC50 value of gemcitabine in ASODN-transfected cell was (0.8±0.2) μmoI/L while that in oligofectamine-transfected control cell (7.3±0.9) μmol/L with a statistically significant difference, hTERT ASODN significantlly increased the gemcitabine-induced apoptosis rate in pancreatic cancer cell. The gemcitabine-induced apoptosis rate in ASODN-transfected cell was 60.28% while that in oligofectamine-transfected control cell 17.34%. Conclusion hTERT antisense oligodeoxynucleotide can increase the sensitivity of pancreatic cancer cell to gemcitabine. The mechanism may be due to the down-regulated expression of hTERT mRNA and telomerase activity.