中文摘要：

目的：构建针对大鼠骨桥蛋白（osteopontin,OPN）基因的小干扰RNA（microRNA,miRNA）绿色荧光蛋白表达载体,并转染大鼠原代皮肤成纤维细胞后,通过半定量RT-PCR法对其干扰效率进行鉴定,从而筛选出最佳的干扰序列。方法：体外化学合成大鼠OPN序列特异性pre-miRNA双链（OPNi-1,OPNi-2及OPNi-3）和非特异性对照双链OPNi-3M,并分别将退火后的双链克隆至线性质粒表达载体pcDNATM6.2-GW/EmGFP-miR。经测序验证后,脂质体介导转染293T细胞进行真核表达验证,并进一步将4种重组质粒经脂质体介导转染大鼠原代皮肤成纤维细胞,半定量RT-PCR法检测各个重组质粒对OPN的干扰效果。结果：经测序验证并转染293T细胞后证明4种重组质粒均构建成功。进一步转染大鼠原代皮肤成纤维细胞后,半定量RT-PCR结果显示OPNi-1、OPNi-2、OPNi-3和OPNi-3M对OPN抑制率分别为49%、35%、21%和2%,而各组间β-actin表达无显著性差异。结论：成功构建针对大鼠OPN的miRNA真核表达载体,其中OPNi-1对大鼠原代成纤维细胞OPN的表达具有最佳抑制率,而对照组OPNi-3M几乎无干扰效果。

英文摘要：

Objective： To construct green fluorescent protein expression plasmids of MicroRNA targeting on the expression of rat osteopontin （OPN）. and to observe the inhibitory effect of the recombinant plasmids on pfimary rat skin fibroblasts through Senti-quantitative RT-PCR. Methods： Chemical synthesis double strands pre-miRNA targeting on rat OPN （OPNi-1, OPNi-2 and OPNi3）and the mock （OPNi-3M） were cloned into pcDNA^TM6.2-GW/EmGFP-miR. After sequencing, the plasmids were transiently transfected into 293T cells separately for expression testing, and then transfected into primary rat skin fihroblasts for identification of inhibitory effect on OPN expression, which was also observeQ782d by semiquantitative RT-PCR. Results： Recombinant plasmids （OPNi-I, OPNi-2. OPNi-3 and OPNi-3M） were confirmed by sequencing and 293T cell transfection analysis. After transfected into primary rat skin fibroblasts, semi-quantitative RTPCR assay showed that OPNi-1, OPNi-2. OPNi-3, and OPNi-3M could suppress the OPN expression by 49% ,35% ,21% and 2% respectively. Conclusion： Recombinant plasmids of MicroRNA targeting on rat OPN were successfully constructed, and the inhibitory effect of OPNi-1 was prominent. In contrast, OPNi-3M could hardly suppress the expression of OPN in primary rat skin fibroblast.