中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (8): 659-664.doi: 10.35541/cjd.20210748

• 论著 • 上一篇    下一篇

CRISPR-Cas9诱导KRT5基因突变的HaCaT细胞对共培养人黑素细胞的影响研究

贾苇雪1    王建波2    罗玲玲1    张媛媛1    王雪3    郭右铭1    孔令卓1    姜祎群4    李诚让1，3   

  1. 1中国医学科学院、北京协和医学院皮肤病医院皮肤科，南京  210042；2河南省人民医院皮肤科，郑州  450003；3江苏省皮肤病与性病学重点实验室，南京  210042；4中国医学科学院、北京协和医学院皮肤病医院病理科，南京  210042
    贾苇雪现在郑州大学第一附属医院皮肤科，郑州  450000
  • 收稿日期:2021-10-14 修回日期:2022-04-28 发布日期:2022-08-02
  • 通讯作者: 姜祎群；李诚让 E-mail:yiqunjiang@qq.com
  • 基金资助:
    江苏省自然科学基金（BK20211027）；河南省自然科学基金（202300410386）

Effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on co-cultured human melanocytes

Jia Weixue1, Wang Jianbo2, Luo Lingling1, Zhang Yuanyuan1, Wang Xue3, Guo Youming1, Kong Lingzhuo1, Jiang Yiqun4, Li Chengrang1,3   

  1. 1Department of Dermatology, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China; 2Department of Dermatology, Henan Provincial People′s Hospital, Zhengzhou 450003, China; 3Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China; 4Department of Pathology, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China
    Jia Weixue  is working at the Department of Dermatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000， China
  • Received:2021-10-14 Revised:2022-04-28 Published:2022-08-02
  • Contact: Jiang Yiqun; Li Chengrang E-mail:yiqunjiang@qq.com
  • Supported by:
    Natural Science Foundation of Jiangsu Province of China （BK20211027）; Natural Science Foundation of Henan Province of China （202300410386）

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摘要： 【摘要】 目的 探究敲减角质形成细胞KRT5基因对共培养黑素细胞黑素含量的影响，以解释屈侧网状色素沉着症（DDD）的色素增加性皮损形成的机制。方法 通过成簇的规律间隔的短回文重复序列-相关蛋白9（CRISPR-Cas9）技术获得KRT5基因杂合突变的HaCaT细胞，将转染非靶向单向导RNA：Cas9蛋白复合物的HaCaT细胞设为对照组，分别与人原代黑素细胞HEMn体外共培养。免疫荧光观察共培养细胞中细胞角蛋白及黑素小体表达；差异胰酶消化获得不同共培养组中黑素细胞，检测细胞内黑素含量。免疫组化检测1例KRT5突变DDD患者皮损和正常对照皮肤中黑素细胞特异性前黑素小体蛋白17（Pmel17）的表达改变。结果 Sanger测序显示，实验组HaCaT细胞KRT5基因第1外显子起始密码子发生杂合突变（c.1delA），对照组HaCaT细胞KRT5基因未发生突变。Western印迹显示，实验组HaCaT细胞KRT5蛋白表达水平（0.60 ± 0.05）显著低于对照组（1.00 ± 0.00，t = 32.38，P = 0.001）。HEMn细胞与实验组HaCaT细胞共培养体系中Pmel17标记的黑素小体较与对照组共培养体系增多，且细胞内黑素含量增加52.5%，差异具有统计学意义（t = -3.48，P = 0.025）。KRT5突变DDD患者皮损组织中Pmel17表达量较正常对照皮肤增加。结论 本研究通过CRISPR-Cas9诱导KRT5基因突变的HaCaT细胞，与黑素细胞在体外建立共培养细胞模型，验证了其对共培养黑素细胞的影响，为进一步研究角质形成细胞与黑素细胞相互作用机制以及皮肤色素异常的发病机制提供了初级研究模型。

关键词: 黑素细胞, 角蛋白细胞, 共同培养技术, 角蛋白5, 生物, 基因修饰, 色素沉着过多, 屈侧网状色素沉着症, CRISPR-Cas9

Abstract: 【Abstract】 Objective To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures （Dowling-Degos disease, DDD）. Methods HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats （CRISPR）-CRISPR-associated protein 9 （Cas9） technology （experimental group）, and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn） separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 （Pmel17） in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results Sanger sequencing showed a heterozygous mutation （c.1delA） at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group （0.60 ± 0.05） than in the control group （1.00 ± 0.00, t = 32.38, P = 0.001）. Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% （t = -3.48, P = 0.025） in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.

Key words: Melanocytes, Keratinocyte, Coculture techniques, Keratin-5, Organisms, genetically modified, Hyperpigmentation, Reticulate pigmented anomaly of the flexures, CRISPR-Cas9