摘要
【目的】观察反义骨调素(OPN)寡核苷酸(AS-ODN)对大鼠肾小管上皮细胞(NRK52E)骨调素mRNA表达及黏附能力的影响;比较AS-ODN的游离形式和经阳离子脂质体(DOTAP)包裹形式的反义抑制效果。【方法】用荧光显微镜观察荧光素标记的AS-ODN在细胞中的分布;将寡核苷酸处理NRK52E细胞48h,用TRIzol试剂提取细胞总RNA;用RNA斑点杂交和RT-PCR检测OPNmRNA的表达;将ODN/DOTAP复合物处理的细胞置于胶原凝胶表面,计算细胞的黏附率。【结果】AS-ODN能转移进入胞核中;AS-ODN处理的细胞OPNmRNA表达水平均较低(P<0.05),而顺义或错义寡核苷酸处理的细胞OPNmRNA水平较高,即使30μmol/L浓度也无抑制作用;游离AS-ODN最低抑制浓度为3.75μmol/L,而AS-ODN浓度为0.5μmol/L的AS-ODN/DOTAP复合物具有良好抑制作用;AS-ODN处理的细胞黏附率降低,而顺义或错义寡核苷酸处理的细胞黏附能力较强。【结论】反义骨调素寡核苷酸能特异地抑制大鼠肾小管上皮细胞OPNmRNA表达和黏附胶原凝胶能力;阳离子脂质体能促进AS-ODN的反义抑制作用。
Objective] To investigate the effects of osteopontin(OPN) antisense oligodeoxynucleotide(AS ODN) on OPN mRNA expression in rat renal epithelial cell line(NRK52E) and adherence of the cells to collagen gel. To compare antisence inhibitory effect of AS ODN /DOTAP(cationic liposome) complex with that of free AS ODN. Fluorecein labeled AS ODN was incubated with NRK52E cells and observed for its intracellular distribution under fluorensence microscopy. When treated with ODN for 48 hours, the cultured NRK52E cells were extracted for total cellular RNA with TRIzol reagent and detected for OPN mRNA expression by RNA dot blot and RT PCR.The ODN/DOTAP complex treated cells were added to collagen gel surface and its adhensive rate was calculatea. AS ODN could incorporate into cell nucleus.AS ODN could inhibit the expression of OPN mRNA regardless of its ODN/DOTAP complex or free ODN(P< 0 05),but mismatch ODN or sense ODN had no inhibitory effects even if at concentration of 30 μ mol/L.Free AS ODN showed little inhibition at concentration of 3.75 μ mol/L ,but AS ODN/DOTAP complex with concentration of 0.5μ mol/L of AS ODN still had significant inhibitory effects.AS ODN treated cells weakly adhered to the collagen gel and easily detached off ,but mismatch ODN or sense ODN treated cells still kept a good adherence.[ Conclusion] Osteopontin antisense oligodeoxynucleotide can specifically inhibit OPN mRNA expression and adhension to collagen gel of NRK52E cells. Cationic liposome can enhance AS ODN antisense inhibitory effect.
出处
《中山大学学报(医学科学版)》
CAS
CSCD
北大核心
2003年第5期439-443,447,共6页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金资助项目(39970704)
广东省自然科学基金资助项目(980058
010763)