Inhibitory effects of ostcopontin antisense oligodeoxynucleotide on osteopontin mRNA expression and adhensive ability of rat renal mcsangial cell / 中华肾脏病杂志

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 ?mol/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 ?mmol/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.