【摘要】 目的研究脂质体介导角蛋白17(K17)反义寡核苷酸对培养人角质形成细胞增殖和K17表达的影响。方法利用脂质体将人工合成的正义、反义及错配K17寡核苷酸基因片段导入体外培养的人角质形成细胞系HaCaT,应用MTT法检测其对HaCaT细胞增殖的影响,以逆转录聚合酶链反应(RT- PCR)检测K17 mRNA水平的变化,并以蛋白质印迹法、免疫荧光细胞化学结合激光扫描共聚焦显微镜检测K17蛋白水平的改变。结果脂质体介导的K17反义寡核苷酸转染HaCaT细胞后,细胞增殖受到明显抑制,同时细胞中K17 mRNA和蛋白的表达明显下降,而正义寡核苷酸组、错义寡核苷酸组及空白对照组均无明显变化。结论应用反义技术封闭K17基因,可以阻遏角蛋白K17基因和蛋白的表达,抑制角质形成细胞的体外生长和增殖能力。更多还原

【Abstract】 Objective To investigate the effects of antisense oligonucleotides of keratin 17 (K17) on the proliferation and expression of K17 in keratinocytes in vitro. Methods The antisense, sense and mismatched oligonucleotides for K17 gene were synthesized and conjugated with lipofectin, respectively. They were subsequently transfected into keratinocyte cell line HaCaT in vitro. Cell proliferation was detected with MTT colorimetric assay. K17 mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR).K17 protein level was detected by Western blot, immuno-fluorescent cytochemistry combined with laser scanning confocal microscopy (LSCM). Results The proliferation of HaCaT cells was inhibited and the expression of K17 gene and protein was markedly decreased after the treatment with antisense rather than with sense and mismatched K17 oligonueleotides. Conclusion Antisense oligonucleotides conjugated with lipofectin might be a useful method to inhibit the proliferation of keratinocytes by inhibiting the expression of K17 mRNA and protein.更多还原