摘要
目的研究骨桥蛋白(OPN)反义寡核苷酸(antisense oligonucleotide,ASODN)抑制人卵巢癌细胞株SKOV3粘附侵袭及诱导凋亡的作用。方法用脂质体将骨桥蛋白ASODN转染卵巢癌细胞SKOV3。实验分3组:对照组,随机序列寡核苷酸(random oligonucleotide,rODN)组和ASODN组。通过荧光定量RT-PCR、流式细胞仪检测法、黏附细胞计数法和Boyden小室法分别测定骨桥蛋白mRNA和细胞表面骨桥蛋白的蛋白表达及癌细胞黏附力、侵袭力的变化。Annexin V染色法、DNA ladder法和Hoechst染色法检测凋亡。结果转染后ASODN组骨桥蛋白mRNA的相对定量比值较对照组显著降低(1.95±0.81 vs 5.26±0.66,P<0.01);流式细胞仪检测骨桥蛋白表达结果显示,转染后平均荧光指数明显降低,细胞表面骨桥蛋白的蛋白表达下降(P<0.01);转染后ASODN组黏附力和侵袭力均较对照组显著下降(黏附细胞百分数值为16.97±1.60 vs 36.14±1.16,穿透聚碳酸酯膜细胞数为44.67±2.08 vs 83.67±4.51,P值均<0.01);经骨桥蛋白ASODN作用后SKOV3细胞发生凋亡。而rODN组上述各指标较对照组无明显变化。结论骨桥蛋白的表达与黏附侵袭能力密切相关,反义寡核苷酸能有效抑制卵巢癌细胞株SKOV3细胞骨桥蛋白基因的表达,从而抑制蛋白质的合成,降低其黏附侵袭能力,并能够诱导凋亡。
Objective To study the effect of osteopontin（OPN） antisense oligonucleotide（ASODN） on the adhesion and nvasion in ovarian cancer cell line SKOV 3 and inducing apoptosis. Methods Three experimental groups were defined as ASODN group,random oligonucleotide （rODN） group and control group. ASODN was transfected into SKOV3 cells by liposome. The expression of osteopontin mRNA and protein were determined by fluorescent quantitative RT-PCR and FCM assay, respectively. The abilities of adhesion and invasion were measured by counting adhesive cells and Boyden chamber test. Apoptosis was investigated by Annexin V/PI staining,DNA ladder and Hoechst staining methods. Results Relative mRNA level of OPN was significantly lower in ASODN group than that in control group （1.95±0.81 vs 5.26±0. 66,P〈 0. 01）. The mean fluorescent index of ASODN group detected by FCM assay was evidently decreased than that of control group （P〈0. 01）. The adhesive and invasive ability of ASODN group decreased more than that of control group （The ratio of adhesive cells was （16.97±1.60） % vs （36.14 ± 1.16） % ,and the number of cells that infiltrated the polycarbonate membrane was 44. 67± 2. 08 vs 83. 67± 4.51,both of P〈0. 01）. Apoptosis appeared after ASODN was transfected into ceils. However, no difference about above-mentioned indexes existed between rODN group and control group. Conclusions The expression of OPN in SKOV3 cells is related with adhesion and invasion. ASODN could effectively inhibit expression of osteopontin gene,inhibit protein synthesis,decrease ability of adhesion and invasion and induce apoptosis.
出处
《实用肿瘤杂志》
CAS
2008年第5期415-420,共6页
Journal of Practical Oncology
关键词
卵巢肿瘤
骨桥蛋白
寡核苷酸类
反义
细胞凋亡
肿瘤侵润
肿瘤标记
生物学
基因表达
ovarian neoplasms
osteopontin
oligonucleotides, antisense
apoptosis
neoplasm invasiveness
tumor markers,biological
gene expression
