中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (4): 224-226.

• 论著 • 上一篇    下一篇

反义寡核苷酸阻遏角蛋白14的表达

陈玉欣, 李巍, 樊建勇, 刘玉峰, 高天文   

  1. 第四军医大学西京医院皮肤科 西安 710032
  • 收稿日期:2003-06-11 出版日期:2004-04-15 发布日期:2004-04-15
  • 通讯作者: 刘玉峰
  • 基金资助:
    中华医学会皮肤科分会科研基金

Downregulation of K14 Genes and Protein Expression by Liposome Conjugated Antisense Oligonucleotides in Keratinocytes

CHEN Yu-xin, LI Wei, FAN Jian-yong, LIU Yu-feng, GAO Tian-wen   

  1. Centre of Dermatology and Venereology of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China
  • Received:2003-06-11 Online:2004-04-15 Published:2004-04-15

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摘要： 目的 研究脂质体介导角蛋白K14反义寡核苷酸(ASODN)阻遏人角质形成细胞K14基因和蛋白的表达及抑制体外增殖活性的效果.方法 人表皮角质形成细胞原代培养,3~10代用于实验,利用脂质体将人工合成的正义、反义及错配K14寡核苷酸基因片段导入角质形成细胞,应用流式细胞仪、逆转录聚合酶链反应(RT-PCR)和免疫组化(SABC)方法检测反义寡核苷酸对角质形成细胞的细胞周期、K14基因和蛋白表达的影响.结果 脂质体介导的K14反义寡核苷酸转染角质形成细胞后,能有效地抑制角质形成细胞中K14基因的表达;48h后可阻遏K14蛋白表达;流式细胞仪检测见反义寡核苷酸处理组细胞周期发生明显改变,G1期细胞百分率上升,S期细胞百分率下降.而正义寡核苷酸1组、错义寡核苷酸组及空白对照组均无此变化.结论 应用反义技术封闭K14基因,可以阻遏角蛋白K14基因和蛋白的表达,并可以抑制体外培养的角质形成细胞的增殖.

关键词: 寡核苷酸类,反义, 角蛋白, 角蛋白细胞, 脂质体

Abstract: Objective To probe into the gene therapy of psoriasis using antisense oligonucleotides to attenuate the expression of K14 gene and protein in keratinocytes and evaluate the inhibitory effects of liposome conjugated antisense oligonucleotides on the proliferation of keratinocytes. Methods The antisense, sense and mismatched oligonucleotides for K14 gene were synthesized and conjugated with lipofectin respectively. Finally they were subsequently transfected into cultured keratinocytes in vitro. The expression of K14 gene was tested by reverse transcription polymerase chain reaction (RT-PCR). The expression of K14 protein was measured by immunohistochemistry. The variation of cell growth cycle was detected by flow cytometry. Results The expression of K14 gene and protein was markedly decreased in keratinocytes treated with K14 antisense oligonucleotides. The cell growth cycle was inhibited effectively by antisense oligonucleotides with lipofection, but not by sense and mismatched oligonucleotides. Conclusions Antisense oligonucleotides conjugated with lipofectin might be a hopeful method to inhibit the proliferation of keratinocytes by inhibiting the expression of K14 mRNA and protein.

Key words: Oligonucleotides, antisense, Keratin, Keratinocytes, Liposomes