转染Livin反义寡核苷酸对人胆管癌QBC939细胞凋亡影响及机制

Study of transfection of Livin antisense oligodeoxynucleotide on apoptosis of QBC939 cells and its mechanism

目的 观察转染Livin反义寡核苷酸（ASODN）对人胆管癌细胞株QBC939细胞Livin mRNA和蛋白表达的抑制作用及其对细胞凋亡的影响.方法 设计合成全硫代磷酸化修饰的Livin ASODN,脂质体介导Livin ASODN转染QBC939细胞,于转染后60h,噻唑蓝（MTT）法检测Livin反义寡核苷酸对QBC939细胞增殖的抑制作用,逆转录-聚合酶链反应（RT-PCR）检测转染前后Livin mRNA表达的变化；细胞免疫荧光化学检测转染前后Livin蛋白的表达变化；膜联蛋白V（Annexin V）-藻红蛋白（PE）流式细胞仪（FCM）法检测转染后细胞凋亡变化.结果 实验分反义（Livin ASODN）组、错义（Livin NSODN）组、脂质体组、空白对照组.转染后60 h,MTT法显示Livin ASODN组能明显促进胆管癌细胞的凋亡（46.41±1.13、3.10 ±0.44、2.66±0.26、0）；RT-PCR显示Livin ASODN组LivinmRNA表达水平明显低于各对照组（54.96 ±3.00、88.31 ±2.50、85.90±0.62、90.36±2.43）（P＜0.05）；细胞免疫荧光化学法显示Livin ASODN组Livin蛋白表达明显低于各对照组（39.05±2.52、8.60 ±3.60、6.37 ±2.71、0）（P＜0.05）；流式细胞仪分析结果显示Livin ASODN组的细胞凋亡明显高于各对照组[（36.98±4.09）％、（3.77±0.25）％、（7.51±0.30）％、（6.83±0.23）％].结论 脂质体介导转染Livin ASODN能特异性抑制QBC939细胞中的Livin基因表达,下调Livin蛋白的表达,抑制胆管癌细胞增殖,并诱导细胞凋亡.

Objective To study the effect of transfeetion of Livin antisense oligodeoxynucleotide （Livin ASODN） on Livin mRNA and Livin protein expression and apoptosis of QBC939 cells. Methods Livin ASODN were transfeeted into cell line QBC939 by LipofeetamineTM 2000. 60 h After transfeetion, to measure Livin mRNA expression by reverse transeriptase polymerase chain reaction （RT-PCR） and Livin protein expression by immunohistoehemistry and confocal laser scanning microscopy; the changes of cell ap optotic were detected by methyl thiazol tetrazolium （MTF）. and the apoptosis of QBC939 cell was detected by Annexin V-PE flow cytometry （FCM） after the transfection. Results The mRNA and protein expres sion level of Livin in ASODN group was significantly lower than that in control group （ P 〈 0. 05 ） （ 54. 96 ± 3.00, 88.31 ±2. 50, 85.90 ±0. 62, 90. 36 ±2. 43） ; the results of MT＇F showed that the rate of cell apoptosis of QBC939 cells were the most obvious at 60 hours after Livin ASODN transfection （46. 41 ± 1.13, 3. 10 ± 0. 44, 2. 66 ±0. 26, 0） （P 〈0. 05）. the apoptosis of QBC939 cell was higher than those in control group af terLivin ASODN transfection [（36.98 ±4.09）%, （3.77 ±0.25）%, （7.51 ±0.3）%, （6.83 ± 0. 23）% ]. Conclusion The transfection of Livin ASODN inhibited Livin gene and protein expression, and Obviously induced apoptosis of Bile duct cancer QBC939 cells, which may be the new target for gene therapy in Bile duet cancer.