中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2008年
10期
741-744
,共4页
刘秀菊%郭其森%张琼%宋现让%柳永蕾%郭琛
劉秀菊%郭其森%張瓊%宋現讓%柳永蕾%郭琛
류수국%곽기삼%장경%송현양%류영뢰%곽침
DNA拓扑异构酶Ⅰ基因%RNAi%小细胞肺癌细胞株H446%拓扑替康%药物敏感性
DNA拓撲異構酶Ⅰ基因%RNAi%小細胞肺癌細胞株H446%拓撲替康%藥物敏感性
DNA탁복이구매Ⅰ기인%RNAi%소세포폐암세포주H446%탁복체강%약물민감성
Top Ⅰ gene%RNA inference%Small cell lung cancer cell line H446%Topetecan%Drug-sensitivity
目的 检测拓扑异构酶Ⅰ(TopI)在小细胞肺癌(SCLC)细胞株中的表达,并探讨其表达水平对拓扑替康(TPT)敏感性的影响.方法 采用Western blot检测TopⅠ在SCLC细胞株H446蛋白水平的表达;应用人工合成并荧光标记的小干扰RNA(siRNA),通过脂质体瞬时转染H446细胞.流式细胞仪检测转染效率;应用定量逆转录聚合酶链反应(RT-PCR)和Western blot检测各干扰序列对TopI在mRNA及蛋白水平的干扰效果,筛选最佳干扰序列;对转染后的细胞株进行药物敏感性实验,观察TopI的表达对TPT敏感性的影响.结果 TOPI基因在H446中有较高表达;siRNA干扰效果满意,转染效率可达86.7%左右;siRNA oligo TopI-892、siRNA oligo TopI-1152、siRNA oHgo TopI-2084和siRNA oligo TOpI-2397等4个干扰序列均抑制H446细胞%pI mRNA的表达,抑制率分别为(95.7 ±1.6)%、(90.8 ±1.6)%、(96.1±2.7)%和(96.3±1.8)%.序列siRNA oligo TopI-2084和siRNA oligo TopI-239r7干扰后,H446细胞TopI蛋白表达明显降低.药物敏感性实验结果显示,相同浓度的TPT对实验组H446细胞的抑制率明显低于转染前及阴性对照的H446细胞(P<0.01).结论 脂质体介导的人工合成siRNA瞬时转染可有效抑制H446细胞的TOPI表达,明显降低细胞对TPT的敏感性.
目的 檢測拓撲異構酶Ⅰ(TopI)在小細胞肺癌(SCLC)細胞株中的錶達,併探討其錶達水平對拓撲替康(TPT)敏感性的影響.方法 採用Western blot檢測TopⅠ在SCLC細胞株H446蛋白水平的錶達;應用人工閤成併熒光標記的小榦擾RNA(siRNA),通過脂質體瞬時轉染H446細胞.流式細胞儀檢測轉染效率;應用定量逆轉錄聚閤酶鏈反應(RT-PCR)和Western blot檢測各榦擾序列對TopI在mRNA及蛋白水平的榦擾效果,篩選最佳榦擾序列;對轉染後的細胞株進行藥物敏感性實驗,觀察TopI的錶達對TPT敏感性的影響.結果 TOPI基因在H446中有較高錶達;siRNA榦擾效果滿意,轉染效率可達86.7%左右;siRNA oligo TopI-892、siRNA oligo TopI-1152、siRNA oHgo TopI-2084和siRNA oligo TOpI-2397等4箇榦擾序列均抑製H446細胞%pI mRNA的錶達,抑製率分彆為(95.7 ±1.6)%、(90.8 ±1.6)%、(96.1±2.7)%和(96.3±1.8)%.序列siRNA oligo TopI-2084和siRNA oligo TopI-239r7榦擾後,H446細胞TopI蛋白錶達明顯降低.藥物敏感性實驗結果顯示,相同濃度的TPT對實驗組H446細胞的抑製率明顯低于轉染前及陰性對照的H446細胞(P<0.01).結論 脂質體介導的人工閤成siRNA瞬時轉染可有效抑製H446細胞的TOPI錶達,明顯降低細胞對TPT的敏感性.
목적 검측탁복이구매Ⅰ(TopI)재소세포폐암(SCLC)세포주중적표체,병탐토기표체수평대탁복체강(TPT)민감성적영향.방법 채용Western blot검측TopⅠ재SCLC세포주H446단백수평적표체;응용인공합성병형광표기적소간우RNA(siRNA),통과지질체순시전염H446세포.류식세포의검측전염효솔;응용정량역전록취합매련반응(RT-PCR)화Western blot검측각간우서렬대TopI재mRNA급단백수평적간우효과,사선최가간우서렬;대전염후적세포주진행약물민감성실험,관찰TopI적표체대TPT민감성적영향.결과 TOPI기인재H446중유교고표체;siRNA간우효과만의,전염효솔가체86.7%좌우;siRNA oligo TopI-892、siRNA oligo TopI-1152、siRNA oHgo TopI-2084화siRNA oligo TOpI-2397등4개간우서렬균억제H446세포%pI mRNA적표체,억제솔분별위(95.7 ±1.6)%、(90.8 ±1.6)%、(96.1±2.7)%화(96.3±1.8)%.서렬siRNA oligo TopI-2084화siRNA oligo TopI-239r7간우후,H446세포TopI단백표체명현강저.약물민감성실험결과현시,상동농도적TPT대실험조H446세포적억제솔명현저우전염전급음성대조적H446세포(P<0.01).결론 지질체개도적인공합성siRNA순시전염가유효억제H446세포적TOPI표체,명현강저세포대TPT적민감성.
Objective To investigate the expressions of Top Ⅰ gene in small cell lung eancer ecll line H446,and explore the influence of Top Ⅰ on the ehemosenaitivity of the cell line to topotecon(TPT). Methods Western blot was performed to detect the Top Ⅰ expression in H446 cells. Lipofectamine 2000 was used for the transient transfection of H446 cells by siRNA, and the transfection efficacy was detected. TopI mRNA was analyzed by quantitative RT-PCR and TopI protein was detected by Western blot to selected effective siRNA. The drug-sensitivity to topotecan (TPT) was evaluated by MTr assay. Results TopI gene was expressed in H446 cells. Lipofectamine 2000 mediated the siRNA effectively (88.67%). Compared with its parental ceils, RT-PCR results showed that TOPI mRNAs in trandected cells were reduced by (95.7±1.6)%, (90.8±1.6)%, (96.1±2.7)% and (96.3±1.8)%, respectively, and decreased significantly at protein level. By MTT essay, the inhibition rate of TPT to H446 cells transfected by siRNA was lower than that of control group at same concentrations (P<0.01 ). Concittsion siRNAs can silence the expression of Top I and decrease the drug-sensitivity of H446 cells to TPT.
