摘要
目的探讨二氢叶酸还原酶（DHFR）基因表达上调对卵巢上皮性癌（卵巢癌）细胞顺铂耐药的影响。方法构建携带DHFR基因的重组慢病毒载体（即重组载体DHFR—pWPI），利用脂质体lipofectamine2000将其转染至卵巢癌细胞系SKOV3细胞中。实验分为3组：转染组（转染重组载体DHFR-pWPI质粒）、阴性对照组（转染慢病毒载体pWPI质粒）、空白对照组（未转染任何质粒）。（1）蛋白印迹（western blot）法检测3组SKOV3细胞中DHFR蛋白的表达；（2）流式细胞仪检测3组SKOV3细胞经不同浓度（2．5、5．0、10．0、20．0μg／ml）顺铂作用不同时间（24、48、72h）后的细胞凋亡率，以及经3组细胞各自的50％抑制浓度（IC50;分别为6．0、4．0、4．9μg／ml，下同）的顺铂作用不同时间（24、48、72h）后的细胞周期比例；（3）高效液相色谱法检测3组SKOV3细胞经不同浓度（4．0、6．0、8．0μg/ml）顺铂作用不同时间（24、48h）后其细胞内顺铂浓度的变化；（4）透射电镜观察3组SKOV3细胞经各自的IC，。的顺铂作用不同时间（24、48、72h）后细胞超微结构的变化。结果本研究成功构建携带DHFR基因的重组载体DHFR—pWPI并转染入SKOV3细胞。（1）westernblot法检测显示，转染组细胞中DHFR蛋白表达水平为10．280±0．009，明显高于阴性对照组及空白对照组（分别为2．050±0．003、3．480±0．003；P〈0．01）。（2）流式细胞仪检测显示，不同浓度（2．5、5．0、10．0、20．0μg／m1）的顺铂作用24、48h后，转染组细胞凋亡率均明显低于阴性对照组和空白对照组（P〈0．05）；而顺铂浓度为5．0、10．0μg／ml作用72h后，转染组细胞凋亡率则明显高于阴性对照组和空白对照组（P〈0．05）。3组细胞经各自IC50的顺铂作用24、48h时，其细胞均以G0／G1期细胞为主，且转染组GJG。期细胞比例明显高于阴性对照组和空白对照组（P〈0.05）；而作用72h时，3组细胞均以GYM、S期细胞为主，且转染组S期细胞比例明显高于阴性对照组和空白对照组（P〈0．01），G2/M期细胞比例明显低于阴性对照组和空白对照组（P〈0.01）。（3j高效液相色谱法检测显示，顺铂浓度为4．0μg／ml作用24、48h后，以及顺铂浓度为6．0μg／ml作用24h后，转染组细胞内的顺铂浓度均明显低于阴性对照组和空白对照组（P〈0．01）；而顺铂浓度为6．0μg／ml作用48h以及8．0μg／ml的顺铂作用24、48h后，转染组细胞内的顺铂浓度均明显高于阴性对照组和空白对照组（P〈0．05）。（4）透射电镜下观察，3组细胞经各自IC50的顺铂作用24、48h后，转染组SKOV3细胞的微丝聚集，线粒体数量及结构改变明显，而空白对照组及阴性对照组细胞微丝少见，线粒体数量减少，但结构改变不明显；3组细胞均出现扩张的内质网，正常的细胞器少见。顺铂作用72h后，转染组细胞未见微丝，核膜几乎完全消失，胞质内有白色囊状物质，线粒体结构基本消失，大部分细胞濒临死亡；而空白对照组及阴性对照组细胞可见微丝排列紊乱，核膜部分消失，核糖体大量聚集，进入明显的坏死阶段。结论本研究采用慢病毒载体系统成功构建了携带DHFR基因的重组慢病毒载体，DHFR基因表达上调后卵巢癌SKOV3细胞的耐药性增加，其耐药性增加可能与微丝的聚集及线粒体的改变有一定的联系。
Objective To investigate the effects of dihydrofolate reductase （DHFR） gene expression up-regulating on cisplatin resistance in epithelial ovarian cancer cell lines. Methods The cDNA length of DHFR gene was amplified by PCR and was connected to lentiviral ＇vector pWPI, the recombinant retroviral vector DHFR-pWPI was infected SKOV3 cells by lipofectamine 2000. The groups included DHFR-pWPI-SKOV3 cell, pWPI-SKOV3 cell and SKOV3 cell group. Western blot was used to detect the expression of DHFR. Flow cytometry was applied to measure the cell apoptosis rate of 3 groups cells in different cisplatin concentrations （2.5, 5.0, 10.0, 20.0 ~g/m]） and at different time period （24, 48 and 72 hours）, and half maximal inhibitory concentration （IC50） treated with cisplatin concentration （6.0, 4.0, 4.9 p,g/ml）. High performance liquid chromatography （HPLC） was applied to test intracellular cisplatin concentration in different cisplatin concentration （4.0, 6.0, 8.0 [xg/ml） at 24 and 48 hours. Transmission electron microscope was used to observe uhrastructural changes cells under ICs0 cisplatin concentration. Results The recombinant plasmid DHFR-pWPI was constructed and then infected into SKOV3 cell successfully. （1） The expression of DHFR detected by western blot in transfection group, was higher than those in the negative control group and blank control group （10.280＋0.009 vs 2.050＋0.003 vs 3.480-＋0.003; P〈0.01）. （2） Treated with cisplatin concentration （2.5, 5.0, 10.0, 20.0 p^g/ml） at 24, 48 hours, the apoptosis rate detected by flow eytometry results shown that they were lower than those in the negative control group and blank control group （P〈0.05）, while treated at the concentration of 5.0 and 10.0 μg/ml for 72 hours, whose apoptosis rate in transfection group was higher than those in the negative control group and blank control group （P〈0.05）. When treated cells under IC50 eisplatin concentration （6.0, 4.0, .4.9μg/ml） at for 24 and 48 hours, the results indicated thatthere were mainly G0/G1 stage cell cycle rate in 3 groups, it was obviously higher in transfection group than those in two control groups （P〈0.05）. However, mainly G2/M, S stage cell cycle rate for 72 hours, and S stage cell cycle rate in transfection group obviously higher than those in two control groups, but GJM stage cell cycle rate were lower （P〈0.01）. （3） After treated with cisplatin concentration （4.0 p^g/ml） for 24, 48 hours and cisplatin concentration （6.0 μg/ml） for 24 hours, the intracellular cisplatin content tested by HPLC method in the transfection group were significantly lower than those in two control groups （P〈0.01）. While, at 6.0 μg/ml of cisplatin concentration for 48 hours and 8.0 μg/ml of eisplatin concentration for 24 and 48 hours, the intraeellular cisplatin content of transfection group were obviously higher than those two control groups （P〈0.05）. （4） Treated with IC,o （6.0, 4.0, 4.9μg/ml） cisplatin concentration at different time to obeserve ultrastruetural changes by transmission electron microscopy. The results shown that the microwire gathered together at 24 and 48 hours, and the number and structure of mitoehondria had obvious change in transfeetion group, while there was rare mierofilament, the number of mitochondria decreased but structure change was not apparent in two control groups. There were appeared expansion of endoplasmic retieulum and had rare normal organelles among three groups. After treated with cisplatin for 72 hours, there were inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in two control groups, and there were rare microfilament in transfeetion group, nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitoehondrial structure disappeared completely, which seems most cells on the verge of death. Conclusion The lentiviral expressing vector harboring human DHFR gene were successfully constructed. When the up-expression of DHFR gene, the drug-resistant in ovarian cancer cell may be increase, which suggest that there were certain contact between resistance increases with mierofilament gathered and the change of the mitochondria.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2015年第11期854-860,共7页
Chinese Journal of Obstetrics and Gynecology
基金
国家自然科学基金（30960404）
国家高技术研究发展计划（012AA02A507）
广西科学研究与技术开发计划（桂科攻1140003A-34）
关键词
卵巢肿瘤
抗药性
多药
抗药性
肿瘤
二氢叶酸还原酶
基因表达调控
Ovarian neoplasms
Drug resistance, multiple
Drug resistance, neoplasm
Dihydrofolate reductase
Gene expression regulation
