【摘要】 目的 观察反义骨调素(OPN)寡核苷酸(AS-ODN)对大鼠肾系膜细胞(1097)骨调素mRNA表达及细胞黏附功能的影响。方法 针对 OPN的cDNA序列设计合成一条与OPN cDNA序列互补的AS-ODN,所有碱基均经硫代修饰并将寡核苷酸与阳离子脂质体(DOTAP)结合。不同浓度寡核苷酸处理1097细胞48h,用Trizol试剂提取细胞总RNA。采用RNA斑点杂交和 RT-PCR技术检测OPN mRNA的表达。采用胶原凝胶黏附法检测细胞的黏附功能。结果 在无血清条件下培养,1097细胞OPN mRNA表达水平很低,加入小牛血清后,其 OPN mRNA表达水平明显上调。反义OPN寡核苷酸处理的1097细胞OPN mRNA表达水平较低,明显低于1640培养基对照组,且具有AS-ODN的浓度依赖性,其最低抑制浓度为2.5μmol/L。而经顺义OPN寡核苷酸或错义OPN寡核苷酸处理的1097细胞OPN mRNA表达水平仍然较高,无明显抑制作用。反义OPN寡核苷酸处理的细胞黏附胶原凝胶的能力较弱,易被洗涤下来;而顺义或错义OPN寡核苷酸处理的细胞黏附凝胶的能力仍然较强。结论 小牛血清能诱导大鼠肾系膜细胞OPN的上调。反义OPN寡核苷酸能特异性地抑制大鼠肾系膜细胞OPN mRNA的表达和对胶原凝胶的黏附能力。更多还原

【Abstract】 Objective To investigate the effects of osteopontin (OPN) antisense oligodeoxynucleotide (AS-ODN)on OPN mRNA expressions and adherence to collagen gel of rat renal mesangial cell line (1097). Methods One AS-ODN complementary chain to rat OPN cDNA sequences was designed and synthesized. All nucleotides of different kinds of oligodeoxynucleotide (including antisense, sense and mismatch sense) were modified with phosphrothioate. All the ODNs were mixed with cationic liposome(DOTAP) respectively. The cultured 1097 cells were incubated with different concentration of ODN at 37t for 48 hours, then total RNA was extracted from cultured cell line with Trizol reagent. OPN mRNA expression was detected with RNA dot blot and RT-PCR. The cell adhesion ability was measured with collagen gel attachment lest. Results OPN mRNA expression of mesangial cells was higherly upregulated in the medium with calf serum compare to that in the medium without serum. AS-ODN could suppress the expression of OPN mRNA in mesangial cells with dose-dependent and its lowest inhibitory concentration was 2. 5 μmol/L, but mismatch ODN or sense ODN have no inhibitory effects on the OPN mRNA expression of mesangial cells even if at a high ODN concentration of 30 μmmol/L. AS-ODN-treated cells weakly adhered to the collagen gel and could be easily detached off. The mesangial cells treated with mismatch ODN or sense ODN still had a good adherent ability to collagen gel. Conclusions Calf serum can induce rat renal mesangial cells higherly expressing OPN mRNA. Osteopontin antisense oligodeoxynucleotide can specifically suppress mesangial cells OPN mRNA expression and adhension to collagen gel.更多还原