RNA干扰对兔角膜基质细胞环氧化酶2与基质金属蛋白酶2表达的抑制作用研究

目的 探讨针对环氧化酶2(COX-2)的RNA干扰(RNAi)对兔角膜基质细胞中COX-2与基质金属蛋白酶2(MMP-2)表达的影响.方法 实验研究.体外培养兔角膜基质细胞,测定阳离子脂质体介导的小干扰RNA(siRNA)在兔角膜基质细胞中的转染率,设计并合成3条针对兔COX-2基因的短发卡RNA(shRNA)1、2、3和1条阴性对照shRNA,利用阳离子脂质体介导转染正常与IL-lα刺激后的兔角膜基质细胞,在IL-lα刺激后6、12、24、48、72 h收集细胞,实时荧光定量聚合酶链反应检测兔角膜基质细胞中COX-2和MMP-2基因的表达变化,并与对照组进行比较.同一时间点各组间比较采用单因素方差分析,处理组两两比较采用q检验.结果 阳离子脂质体能够有效介导siRNA转染体外培养的兔角膜基质细胞,转染率可达70%～80%.IL-lα刺激后兔角膜基质细胞中COX-2与MMP-2 mRNA的表达较空白对照组显著升高;在不同时间点,生物合成的3条针对COX-2的靶向shRNA中shRNA-2能显著抑制IL-α刺激后的兔角膜基质细胞中COX-2、MMP-2 mRNA表达,抑制率最高可分别达83.04%、73.69%,与单纯IL-lα刺激组相比差异均有统计学意义(q=24.03,P=0.00;q=14.76,P=0.00);而shRNA-1、shRNA-3、阴性对照shRNA转染组与单纯IL-lα刺激组相比,COX-2 mRNA的表达差异无统计学意义(F=0.02,P=0.99),MMP-2 mRNA的表达差异也无统计学意义(F=0.02,P=0.98).结论 针对COX-2的RNAi能够有效抑制IL-lα诱导后的兔角膜基质细胞中COX-2与MMP-2的表达.

RNA interference inhibits expression of cyclooxygenase-2 and matrix metalloproteinase-2 in rabbit corneal stromal cells

Objective To research the effect of short haipin RNA (shRNA) targeting cyclooxygenase-2 (COX-2) on the expression of COX-2 and MMP-2 in rabbit corneal stromal cells in vitro.Methods It was a experimental study.Rabbit comeal stromal cells were cultured in vitro.The transfection efficiency of siRNA mediated by HiperFect Transfection Reagent was determined in rabbit comeal stromal cells in order to optimize the transfection condition.shRNAs (shRNA-1,2,3) specific for COX-2 and one negative control nonspecific shRNA were designed,then were transfected by HiperFect Transfection Reagent into both normal rabbit corneal stromal cells and those which were stimulated by IL-lα.6,12,24,48,72 hours after IL-lα added,cells were collected for Real-Time PCR to detect the gene expression of COX-2 and MMP-2.then the results were compared with those of control group.The data of all groups at the same Same time was analyzed by one-factor analysis of variance,and the comparison of any two groups was carried out by q-test. Results The siRNA mediated by HiperFect Tmmfection Reagent can be transfected efficiently into rabbit corneal stromal cells in vitro,and the maximum efficiency was 70%-80%.The expression of COX-2 and MMP-2 mRNA after IL-lα stimulated was much higher than that of blank group.shRNA-2 can significantly inhibit the expression of COX-2 and MMP-2 mRNA in corneal stromal cells stimulated with IL-lα The level of COX-2 and MMP-2 was reduced 83.04% and 73.69% respectively,compared with the expression of single IL-lα stimulated group,the difference had statistical significance (q=24.03,P=0.00;q=14.76,P=0.00).The difference of COX-2 mRNA among transfection groups of shRNA-1,BhRNA-3,negative control shRNA and single IL-lα stimulated group had no statistical significance (F=0.02,P=0.99).The difference of MMP-2 mRNA among those groups also had no statistical significance (F=0.02,P=0.98).Conclusion The RNA interference targeting COX-2 can effectively inhibit the expression of COX-2 and MMP-2 in IL-lα stimulated rabbit comeal stromal cells in vitro.