| PURPOSE: To research the effect of short haipin RNA (shRNA) designed forcyclooxygenase-2 (COX-2) on the expression of COX-2 and MMP-2 in corneal stromalcells of rabbits in vitro.METHODS: Corneal stromal cells of rabbits were cultured in vitro. The transfectionefficiency of siRNA mediated by HiperFect Transfection Reagent was determined incorneal stromal cells of rabbits in order to optimize the transfection condition. Accordingto the COX-2 gene of rabbits,three shRNAs and one negative control shRNA weredesigned and synthesized. The corneal stromal cells of rabbits were inoculated on 24-holecultural plates and divided into 6 groups(A,B,C,D,E,F) at random. Each groupcontained 10 holes and the number of inoculated cells in each hole was about 6α10~4.Group A was blank and the rest 5 were experimental groups. 25ng of IL-1αwas addedinto culture solution per milliliter in group B 36 hours after inoculation. The attenuatedmixture of 3.0μl HiPerFect Transfection Reagent and 25nM different shRNA (shRNA-1,shRNA-2,shRNA-3 and negative control shRNA) was added into groupC,D,E,Frespectively 24 hours after inoculation each hole which contained 0.5ml DMEM free ofblood serum.12 hours after inoculation 25ng of IL-1αwas added into culture solutionper milliliter. 6,12,24,48,72hours after IL-1αadded,two holes of corneal stromal cellsin each group were selected at random and the RNA was extracted for Real-Time PCR todetect the gene expression of COX-2 and MMP-2 in corneal stromal cells,then the resultswere compared with those of control group.RESULTS: The siRNA mediated by HiperFect Transfection Reagent can be transfectedefficiently into corneal stromal cells of rabbits in vitro, the transfection efficiency canreach 70%-80%.The expression of COX-2 and MMP-2 mRNA after IL-1αstimulatedwas much higher than that of blank group,and the level improved with time extended.Among three shRNAs designed for COX-2, shRNA-2 can significantly inhibit theexpression of COX-2 and MMP-2 mRNA in corneal stromal cells stimulated with IL-1α,inhibition rate can reach 83.04%,73.69% respectively ,compared with the expression ofsingle IL-1αstimulated group, the difference had statistical significance (P＜0.01). Thedifference between transfection groups of shRNA-1,shRNA-3 and single IL-1αstimulated group had no statistical significance (P＞0.05). The difference of COX-2 andMMP-2 mRNA expression between the negative control and single IL-1αstimulatedgroup had no statistical significance (P＞0.05). CONCLUSIONS:The RNA interference targeting COX-2 can effectively inhibit theexpression of COX-2 and MMP-2 in IL-1 a stimulated corneal stromal cells of rabbits invitro. |
