摘要
目的探讨顺铂作用于叶酸结合蛋白（FOLRl）基因表达上调的卵巢上皮性癌（卵巢癌）细胞系SKOV3细胞后对其生物学功能的影响。方法将FOLRl基因与pWPI质粒进行慢病毒包装，构建重组质粒pWPI—FOLRl并转染人SKOV3后得到pWPI—FOLR1-SKOV3细胞，作为重组质粒转染组；以同样方法将pWPI质粒转染入SKOV3后得到pWPI—SKOV3细胞，作为空载体转染组；并设立未转染的SKOV3细胞为空白对照组，采用逆转录（RT）一PCR技术和蛋白印迹法分别检测3组细胞中FOLRlmRNA和蛋白的表达。采用四甲基偶氮唑蓝（MTT）比色法，检测并绘制3组细胞的生长曲线，检测3组细胞对JJl^13的敏感性[以50％抑制浓度（IC50）表示，选择重组质粒转染组的IC50作为下一步实验中顺铂浓度的基准]，并检测不同浓度（分别为0．5×IC。1×IC。2×IC。即分别为1．8、3．6、7．2μg/ml；下同）顺铂作用不同时间（分别为24、48、72h，下同）后3组细胞生长的抑制率；采用流式细胞仪检测不同浓度顺铂作用不同时间后3组细胞的凋亡率，及不同浓度顺铂作用48h后3组细胞的细胞周期比例；高效液相色谱法检测同一浓度（3．6μg/ml）顺铂作用48h后3组细胞内N$t3的浓度。结果RT—PCR技术和蛋白印迹法分别检测显示，重组质粒转染组细胞中有FOLRlmRNA和蛋白的表达，而空载体转染组、空白对照组细胞中无FOLRlmRNA和蛋白的表达。MTT比色法检测显示，重组质粒转染组细胞生长速度最快、对顺铂最敏感（其IC50最低，为3．6μg／ml）、相同作用条件（包括顺铂的浓度和作用时间）下其细胞生长的抑制率最高，分别与空载体转染组、空白对照组比较，差异均有统计学意义（P〈0．05）；而空载体转染组与空白对照组比较，差异无统计学意义（P〉0．05）。流式细胞仪检测显示，3组细胞的凋亡率随着顺铂浓度（分别为1．8、3．6、7．2肛
Objective To explore biological effects of up-regulated expression of transfected FOLR1 gene on SKOV3 cell lines following action by cisplatin（DDP）. Methods Three groups of cells originated from the same SKOV3 cell line were used in this research, including the SKOV3 cell line （blank control） , the cell line transfected with lentiviral pWPI plasmid （ no-load control） and the cell line transfected with FOLR1 gene via lentiviral pWPI plasmid （experimental group）. Next, the mRNA and protein expression of FOLR1 gene in the three groups were detected by reverse transcription （RT）-PCR and western blot, respectively. Methyl thiazolyl tetrazolium （MTT） assay was used to analysis cells growth curve and identify their sensitivity to cisplatin,and their half inhibition concentration（ ICs0 ） values were calculated. Based on the ICs0 value （3.6 Ixg/ml） in the experimental group, different levels of cisplatin concentration （0. 5 x ICs0, 1 x ICs0,2 x ICs0, respectively） were administered to the three groups of cells, and the inhibition rates, apoptosis rates as well as apoptosis proportion of each group after 24, 48, 72 hours were further recorded. Finally, the residual cisplatin concentrations in the three group cells acted successively by 1 x ICs0 cisplatin for 48 hours were measured by high performance liquid chromatography（HPLC）. P value less than 0. 05 were defined as statistically significant. Results RT-PCR and western blot detection showed that stable mRNA and protein expression of the FOLR1 gene in the experimental group while the other two groups were not. MTI＂ assay demonstrated that higher cell growth rate, sensitivity to cisplatin（ ICs0 = 3.6 p,g/ml） and inhibition rate in the experimental group compared with those in the other two groups （ P 〈 0. 05 ）, which showed no significance in intergroup comparison（ P 〉 0. 05 ）. Flow cytometry showed apoptosis rates among three groups increased with higher cisplatin concentrations and longer action duration in dosage
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2013年第9期676-682,共7页
Chinese Journal of Obstetrics and Gynecology
基金
国家自然科学基金（30960404）
