Cathepsin K基因siRNA的筛选及其质粒载体的构建

Experimental Study on siRNA Screening of Cathepsin K Gene and Construction of Its Plasmid Vector

目的筛选出对人Cathepsin K(CTSK)基因抑制效率最高的1对siRNA,并通过质粒载体表达该siRNA,为进一步抑制去分化人软骨细胞中CTSK的表达奠定基础。方法针对Cathepsin K基因设计4个靶位点,并根据靶位点合成4对siRNA,并转染人软骨细胞。通过Real-timePCR检测4对siRNA中抑制效率最高的1对,与线性化的PGCsilencer-Hl/Neo/GFP连接、转化、扩增与纯化质粒。结果4对siRNA转染软骨细胞后72h,通过对照FITC-siRNA的绿色荧光观察,siRNA的转染效率达到70%～80%。Real-time PCR检测每对siRNA的CTSK量与对照组比的百分率得到其抑制效率,分别为:第1对的比值大于对照组(无抑制作用),第2对47.5%,第3对53.1%,第4对67.3%(抑制效率最大)。成功构建出pGCsilencerTMH1/Neo/GFP/CTSK RNAi载体,经测序证实了克隆的RNAi打靶序列100%正确。结论成功构建了CTSK基因的siRNAs表达质粒,为进一步研究抑制该基因表达对延缓软骨细胞的去分化过程并促进其成软骨能力奠定了基础。

Objective To screen out the most efficient pair of siRNA will ability of inhibiting the human Cathepsin K （CTSK） gene, and through plasmid vector expressing the siRNA, further depress the dedifferentiation of human chondrocytes. Methods Four pairs of synthetic siRNA were designed and based on target sites of Cathepsin K gene, and transfected human chondrocytes. Detection of four pairs to select a pair of siRNA were detected by Real-time PCR to select a pair of the most efficient. It was connected with linear oriented PGCsilencer -HI/Neo/GFP, transformed, amplified and purified. Results The four pairs of siRNA transfected chondrocytes 72 hours later, by comparing the FITC-siRNA for green fluorescent observation, siRNA transfection efficiency reached 70%-80%. Real-time PCR detection of each pair of siRNA can obtain inhibition efficiency, respectively, the ratio of the first pair is greater than that of the control group, there was no inhibitory effect, and the ratio is 47.5%, 53.1% and 67.3% in the second, third and fourth pain siRNA respectively. A pGCsilencerTM H1/Neo/GFP/CTSK RNAi vector were constructed successfully, the cloning of RNAi targeting sequence of correct was confirmed. Conclusion siRNAs of CTSK gene expression plasmid was constructed. It may play an important role on inhibition of the CTSK gene expression of chondrocytes to slow down the process of dedifferentiation and promote ability of cartilage formation.