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【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 antisense osteopontin (OPN) oligonucleotide (AS-ODN) on osteopontin mRNA expression and adhesion in rat renal tubular epithelial cells (NRK52E) Antisense inhibition with cationic liposome (DOTAP) coated forms. 【Methods】 The distribution of fluorescein-labeled AS-ODN in cells was observed by fluorescence microscopy. NRK52E cells were treated with oligonucleotides for 48h, and the total RNA was extracted by TRIzol reagent. The expression of OPN mRNA was detected by dot blot hybridization and RT-PCR The cells treated with ODN / DOTAP complex were placed on the surface of collagen gel to calculate the cell adhesion rate. 【Results】 The expression of OPN mRNA in AS-ODN-treated cells was lower than that in AS-ODN transfected cells (P <0.05), while the expression of OPN mRNA in AS-ODN-treated cells was higher The concentration of AS-ODN was 3.75μmol / L, while AS-ODN / DOTAP complex with AS-ODN concentration of 0.5μmol / L had a good inhibitory effect. AS-ODN-treated cells Adhesion rate decreased, while Shunyi or missense oligonucleotide treatment of cell adhesion ability. 【Conclusion】 Antisense osteopontin oligonucleotide can specifically inhibit the expression of OPN mRNA and collagen gel in rat renal tubular epithelial cells. Cationic liposomes can promote antisense inhibition of AS-ODN.