靶向角蛋白17基因的小干涉RNA对角质形成细胞增生与凋亡的影响  

The Effects of siRNA Targeting Keratin 17 on the Proliferation and Apoptosis of Cultured Keratinocytes in Vitro

作　　者：王岩[1] 王刚[1] 刘玉峰[1] 孙林潮[1] 沈柱[1] 

机构地区：[1]第四军医大学西京医院皮肤科,陕西西安710032

出　　处：《中国皮肤性病学杂志》2006年第4期201-203,共3页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金课题(No.30371650)

摘　　要：目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。Objective To silence the expression of the human keratin 17 by RNA interference （RNAi） technology, and observe the effects on the cultured keratinocytes in vitro. Methods The mammalian expression vector, psilencer3.1-H1 neo was used for expression of siRNA in human keratinocyte line, HaCaT. The synthetic oligonucleotides targeting K17 were annealed and cloned into the vector to transfect HaCaT,which expressing K17. Western blot studies and RT-PCR assay were testified to observe the changes of the protein and mRNA. level. The cell proliferation and apoptosis were detected by using flow cytometer and electron microscopy. Results K17 RNAi expression vector was successfully constructed and named psilencer3, 1/K17. Compared with that of the control group, the cell proliferation of transfected group was inhibited and apoptosis was higher. Conclusion The expression of K17 has a significant effect on the proliferation and apoptosis of keratinecytes. The siRNA targeting keratin 17 can inhibit its proliferation and induce the apoptosis.

关 键 词：角蛋白17 角质形成细胞 RNA 干涉 银屑病 

分 类 号：R392.13[医药卫生—免疫学]