局部寡核苷酸诱导小鼠毛囊角蛋白17基因改变的基因治疗研究

中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (4): 189-193.

• 论著 • 下一篇

局部寡核苷酸诱导小鼠毛囊角蛋白17基因改变的基因治疗研究

范卫新¹, Kyonggeun Yoon²

1. 南京医科大学第一附属医院皮肤科 210029;
2. Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, USA

收稿日期:2003-04-13 出版日期:2004-04-15 发布日期:2004-04-15
基金资助:
国家自然科学基金资助课题(30170860)

In vivo Alteration of the Keratin 17 Gene in Hair Follicles by Oligonucleotide-directed Gene Targeting

FAN Wei-xin¹, Kyonggeun Yoon²

Dept. Of Dermatology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China

Received:2003-04-13 Online:2004-04-15 Published:2004-04-15

摘要/Abstract

摘要： 目的研究体内角质形成细胞基因修改的可能性.方法通过RNA-DNA寡核苷酸(RDO)或单链DNA寡核苷酸(ssODN)局部皮肤注射的方法,诱导毛囊角蛋白17(K17)(R94P)显性突变.注射的K17A-RDO或K17A-ssODN含有一个单碱基错配(CGC到CCC),从而引起该氨基酸的替换.注射的混合物由上述寡核苷酸和阳离子脂质体组成.分别于小鼠出生后第2天和第5天进行皮内注射.第8天取皮肤活检.结果组织学检查发现注射K17A-RDO的7只小鼠中有5只和注射K17A-ssODN的5只小鼠全部有毛发生长明显异常.其主要表现为毛囊内毛干在皮脂腺水平扭曲盘绕或断裂,个别毛囊毛球部碎裂,黑素颗粒滴落到周围真皮中,有些切片中还可见表皮囊肿.注射部位生长期Ⅳ～Ⅵ期毛囊数减少50%.阴性对照组均未显示明显毛发生长异常.从组织切片上分离异常毛囊并提取DNA,PCR扩增后DNA测序和非限制性内切酶酶切证实,K17基因由正常的CGC变成了CCC.结论局部注射K17A-RDO或K17A-ssODN以导致特异性靶基因K17的定点突变,引起可见的毛发生长异常.

关键词: 角蛋白, 碱基错配, 点突变, 毛囊, 动物替代试验

Abstract: Objective To investigate the possibility of gene correction in keratinocytes. Methods Using intradermal injection of a chimeric RNA-DNA oligonucleotide (RDO) or a single stranded oligonucleotide (ssODN) into murine skin, we attempted to make a dominant mutation (R94P) in the conserved α-helical domain of K17. Both K17A-RDO and K17A-ssODN contained a single base mismatch (CGC to CCC) to alter the normal K17 sequence to cause an amino acid substitution. The complexes consisting of oligonucleotides and cationic liposomes were injected into C57BL/6 murine skin on day 2 and day 5 after birth. Skin biopsies were taken on day 8 after birth. Results Histological examination of skin biopsies on day 8 from mice (5/7 for K17A-RDO and 5/5 for K17A-ssODN) showed consistent twisted hair shafts or broken hair follicles at the sebaceous gland level and an occasional rupture of hair bulb and epidermal cyst-like changes. In the injected area, the number of full anagen hair follicles decreased 50%. Injection of the control oligonucleotide, identical to K17A-RDO but containing no mismatch to the normal sequence, did not result in any detectable abnormality (n=5). The genomic DNA isolated by dissection of hair follicles from slides, PCR amplified and subjected to Aci I digestion, showed that the target gene was mutant from CGC to CCC. Conclusions Our results indicate that intradermal injection of the K17A-RDO or K17A-ssODN affected the hair growth and morphology.

Key words: Hair follicle, Animal testing alternatives, Keratin, Base pair mismatch, Point mutation

范卫新, Kyonggeun Yoon. 局部寡核苷酸诱导小鼠毛囊角蛋白17基因改变的基因治疗研究[J]. 中华皮肤科杂志, 2004, 37(4): 189-193.

FAN Wei-xin, Kyonggeun Yoon. In vivo Alteration of the Keratin 17 Gene in Hair Follicles by Oligonucleotide-directed Gene Targeting[J]. Chinese Journal of Dermatology, 2004, 37(4): 189-193.

参考文献

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局部寡核苷酸诱导小鼠毛囊角蛋白17基因改变的基因治疗研究

In vivo Alteration of the Keratin 17 Gene in Hair Follicles by Oligonucleotide-directed Gene Targeting

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