人增生性瘢痕成纤维细胞IKKβ特异性siRNA表达载体构建及表达

Screening and expression of specific SiRNA targeting IKKβ in human hypertrophic scar fibroblasts and construction of IKKβ-siRNA expression vector

目的:体外合成并筛选人增生性瘢痕成纤维细胞特异性IKKβ-siRNA,构建其表达载体并检测其表达.方法:化学合成3条IKKβ-siRNA,分别转染体外培养的人增生性瘢痕成纤维细胞,采用RT-Q-PCR筛选出特异性IKKβ-siRNA,并采用PU6质粒构建载体,RT-Q-PCR检测其表达.结果:合成的3条IKKβ-siRNA链中,仅第二条有明显抑制IKKβ的mRNA表达的作用,并通过酶切鉴定和测序结果证明成功构建IKKβ-siRNA载体,经转染靶向IKKβ-siRNA的成纤维细胞,IKKβ的表达在转染后24h及48h均受到抑制.结论:应用RT-PCR可筛选出针对人增生性瘢痕成纤维细胞的特异性IKKβ-siRNA;可构建具有干扰效果的IKKβ-siRNA表达载体,转染后可以降低IKKβ的表达.

AIM：To explore the effects of different siRNAs on the expression of IKKβ mRNA and to construct the specific IKKβ-siRNA vector of human hypertrophic scar fibroblasts. METHODS：Three IKKβ-siRNAs （L1,L2 and L3） were transcribed in vitro and then transferred to cultured human hypertrophic scar fibroblasts. RT-PCR was performed to evaluate the levels of IKKβ mRNA in the transferred cells. The specific IKKβ-siRNAs plasmid vector was constructed according to the screened IKKβ-siRNA siRNAs. RESULTS：The expression of IKKβ-siRNA mRNA of fibroblasts transferred with only L2 reduced significantly. Double enzyme digestion and sequence detection showed that the plasmid vector of siRNA specific for IKKβ was successfully constructed. The expression of IKKβ was significantly suppressed at both mRNA and protein levels after transfection. CONCLUSION：Specific IKKβ-siRNA of human hypertrophic scar fibroblasts can be screened out by RT-PCR. The IKKβ-siRNA expression vector of potential interference ability for target tissue can be constructed. The expression of targeting IKKβ-siRNA of fibroblasts transferred reduced significantly.