目的 探讨腺病毒介导的RNA干扰ERCC1基因表达对卵巢癌顺铂化疗的增敏作用。方法 采用直接感染法、台盼蓝活细胞计数、MTT、Hoechst染色法及流失细胞仪检测等方法,绘制SKOV3细胞生长曲线及倍增时间,检测SKOV3细胞感染重组病毒前后其顺铂的IC50值、细胞凋亡形态及细胞各周期的分布。结果 (1)顺铂浓度与SKOV3细胞抑制率呈线性正相关,相关系数r=0.9905(P<0.05),顺铂IC50值为(7.76±0.37)μg/ml;腺病毒载体介导的RNA干扰ERCC1基因表达后,SKOV3细胞的生长受到一定程度的抑制;(2)与对照组相比,梯度浓度的重组腺病毒感染后,SKOV3细胞对顺铂的敏感性分别增加了9.4%、16.4%、23.2%、33.5%,呈剂量依懒性;(3)梯度滴度重组腺病毒表达载体联合顺铂作用于SKOV3细胞株,细胞凋亡显著增高;(4)重组腺病毒表达载体感染并联合顺铂用药,G1期细胞比例减小,S期细胞比例增大,细胞凋亡率进一步增高。结论 重组腺病毒表达载体可以通过诱导肿瘤细胞凋亡、调节肿瘤细胞周期分布等途径增加SKOV3细胞对顺铂的敏感性。
Abstract
Objective To study the enhancement of sensitivity of DDP in ovary carcinoma by expressions of adenovirus-mediated RNA that interferes with ERCC1 genes.Methods SKOV3 cells were infected by direct infection methods and with recombinant adenovirus. Taipan blue dye, MTT, Hoechst staining and flow cytometer were used to draw SKOV3 cell growth curves and doubling time. DDP’sIC50, apoptosis morphology and the cell cycle distribution of ovary carcinoma cell line SKOV3 before and after the recombinant adenovirus infection were detected.Results (1) There was a linear positive correlation between the concentration of DDP and the cell inhibition rate of SKOV3, and the correlation coefficient r=0.9905(P<0.05), while DDP’sIC50 was (7.76±0.37)μg/ml.Ad-HTERT-ERCC1-siRNA had some inhibitory effect on SKOV3.(2)Drug sensitivity test showed that apoptosis of SKOV3 cells increased with the infectious viral titer and the concentration of DDP, and that cell survival rate significantly decreased in a dose-dependent manner.(3)As the infectious viral titer and the concentration of DDP were increased, the cell apoptosis was significantly elevated.(4)The recombinant adenovirus expression vector could increase S stage and cell apoptosis, but decrease G1 stage predominantly.Conclusions The recombinant adenovirus expression vector can effectively enhance DDP’s sensitivity to SKOV3 by inducing the apoptosis of tumor cells and regulating the cycle distribution of tumor cells.
关键词
卵巢癌 /
ERCC1基因 /
RNA干扰 /
重组腺病毒表达载体
Key words
ovarian carcinoma /
ERCC1 gene /
RNA interference /
recombinant adenovirus expression vector
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