摘要
目的探讨ATP7B基因反义寡核苷酸(antisense oligodeoxynucleotide,ASODNs)片段对卵巢癌SKOV3ip1细胞顺铂敏感性的影响。方法人工合成包含ATP7B mRNA翻译起始位点的ASODNs片段及作为对照的正义寡核苷酸(sense ol-igodeoxynucleotide,SODNs)片段。脂质体lipofectamine2000(LF)将ASODNs及SODNs转染卵巢癌SKOV3ip1细胞后,用RT-PCR法、流式细胞测定法、Western blot及MTT法检测SKOV3ip1细胞转染后,ATP7B基因mRNA、蛋白表达及半数抑制浓度(IC50)的变化。结果SKOV3ipl细胞以及转染ASODNs后,ATP7B与β-actin mRNA的光密度比值分别为0.831±0.06和0.203±0.07(P<0.01)。流式细胞法检测细胞内ATP7B蛋白的荧光强度由转染前的79.42±4.04下降到50.87±5.47(P<0.05)。Western blot检测SKOV3ipl细胞以及转染ASODNs后,ATP7B与actin蛋白的光密度比值分别为2.60±0.44和1.01±0.06(P<0.01)。MTT检测IC50由转染前的(126.63±12.06)μmol/L下降为(80.90±20.09)μmol/L(P<0.01)。而转染SODNs及LF的SKOV3ip1细胞中,上述指标无统计学差异(P>0.05)。结论ATP7B基因反义寡核苷酸能显著抑制卵巢癌SKOV3ip1细胞ATP7B mRNA和蛋白表达,并能增加SKOV3ip1细胞对顺铂的敏感性。
Objective.. To investigate whether antisense oligodeoxynucleotides （ASODNs） of ATP7B gene could potentiate the sensitivity of ovarian carcinoma SKOV3ipl cells to cisplatin. Methods： The artificially synthesized ATP7B ASODNs included the translation initiation site. They were transfected into SKOV3ipl cells by lipofectamine 2000（LF）. The sense oligodeoxynucleotides （SODNs） were as control. ATP7B mRNA and protein expression were detected by RT PCR, flow cytometry （FCM） and Western blotting. The cell viability was assessed by MTT assay. Results：After transfected with ASODNs, the optical density ra- tios of ATP7B mRNA to β-actin decreased from 0.83±0.06 to 0.20±0.07 （ P 〈0.01）. The intracellular fluorescence intensi- ties decreased from 79.42±4.04 to 50. 87±5.47 （P〈0.05）. Western blot showed that the optical density ratios of ATP7B protein to β-actin decreased from 2.60±0.44 to 1.01±0.06 （ P 〈0.01 ）. The ICs0 value of cisplatin decreased from （126.00± 12.06） to （80.90±20.09）±mol/L （ P 〈0.01）. No significant changes could be observed in other cells transfected with SODNs and LF only. Conclusion： ASODNs of ATP7B can significantly inhibit the expressions of ATP7B at mRNA and protein levels and increase the sensitivity of ovarian cancer SKOV3ipl cells to cisplatin.
出处
《肿瘤》
CAS
CSCD
北大核心
2006年第1期28-31,共4页
Tumor
基金
上海市卫生局青年基金资助项目(编号:024Y06)
