摘要
背景:近年来的研究表明核转录因子κB通过调控多种基因的表达,参与细胞生长、发育、凋亡等多种生理功能,并在细胞恶性转化、纤维化等方面起重要作用。核转录因子κB抑制蛋白激酶(inhibitor of kappa B kinase,IKK)β是核转录因子κB激活通路中的关键激酶,被抑制后可以阻断核转录因子κB的活化过程,通过siRNA干扰沉默IKKβ基因,下调核转录因子κB。目的:体外合成并筛选人瘢痕疙瘩成纤维细胞特异性IKKβ-siRNA,并构建其表达载体。设计、时间及地点:细胞观察实验,于2008-04/2008-10在解放军广州军区总医院中心实验室完成。材料:细胞来源为珠江医院整形外科25～40岁患者切除的瘢痕疙瘩标本,患者知情同意。方法:化学合成3条IKKβ-siRNA,分别转染体外培养的人瘢痕疙瘩成纤维细胞,采用反转录-聚合酶链反应技术检测所合成的SiRNA对IKKβ表达的抑制,筛选出特异性IKKβ-siRNA,并采用PU6质粒构建载体。主要观察指标:①RT-Q-PCR检测结果。②载体基因连接产物酶切鉴定。③重组质粒测序鉴定。结果:经实时定量反转录-聚合酶链反应检测,合成的3条IKKβ-siRNA链中,IKKβ-siRNA-2有明显抑制IKKβmRNA表达的作用,IKKβ-siRNA-1、IKKβ-siRNA-3干扰效果不明显。并通过酶切鉴定和测序结果证明成功构建IKKβ-siRNA载体。结论:应用反转录-聚合酶链反应可筛选出针对人瘢痕疙瘩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。
BACKGROUND： Studies revealed that nuclear factor κB （NF-κB） participates in cell growth, development and apoptosis via regulating the expression of multiple genes, which plays an important role in ceils malignant transformation or fibration. Inhibitor of κB kinase （IKK） is essential for inflammatory cytokine-induced activation of NF-κB, blocks NF-κB-mediated inflammatory responses, and down regulates NF-κB by interfere with IKKβ gene through siRNA. OBJECTIVE： To screen the effects of different siRNAs on the expression of IKKβ mRNA and to construct the specific IKKβ-siRNA vector of human keloid fibroblasts. DESIGN, TIME AND SETTING： The cytology experiment was performed at the Central Laboratory of Guangzhou Military Area Command of Chinese PLA from April 2008 to October 2008. MATERIALS： Cells were harvested from excisional keloid of patients at the Department of Plastic Surgery, Zhujiang Hospital. Informed consent was obtained from each patient. METHODS： Three IKKβ-siRNAs were transcribed in vitro and then transferred to human keloid fibroblasts. RT-PCR was performed to evaluate the levels of IKKβ mRNA in both transferred cells. The specific plasmid vector of IKKβ-siRNA was constructed. MAIN OUTCOME MEASURES： Results of RT-Q-PCR; identification of gene product; identification of recombinant plasmid. RESULTS： Realtime RT-PCR showed that the IKKβ -siRNA-2 inhabited the expression of IKKβ-siRNA mRNA of fibroblasts, and the other disturbances were not significant, The plasmid vector of sIRNA specific for IKKβ was proved to be constructed successfully by double enzyme digestion and detecting its sequence. CONCLUSION： Specific IKKβ -siRNA of human keloid fibroblasts can be screened by RT-PCR. The IKKβ-siRNA expression vector with potential interference ability for target tissue can be constructed.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第20期3920-3923,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
