中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (2): 95-101.doi: 10.35541/cjd.20210266

• 论著 • 上一篇    下一篇

Spink5基因条件性敲除小鼠模型的构建及表型分析

阎诗    周小英    罗晓燕    任发亮    江为    牛琳琳    王华    白晓明   

  1. 重庆医科大学附属儿童医院皮肤科  儿童感染免疫重庆市重点实验室  国家儿童健康与疾病临床医学研究中心  儿童发育疾病研究教育部重点实验室，重庆  400014
  • 收稿日期:2021-03-30 修回日期:2021-12-05 发布日期:2022-01-27
  • 通讯作者: 白晓明；王华 E-mail:XiaomingBai@CQMU.edu.cn; huawang@ hospital.cqmu.edu.cn
  • 基金资助:
    国家自然科学基金（81703124、81801637）

Construction of a Spink5 conditional knockout mouse model and analysis of its phenotype

Yan Shi, Zhou Xiaoying, Luo Xiaoyan, Ren Faliang, Jiang Wei, Niu Linlin, Wang Hua, Bai Xiaoming   

  1. Department of Dermatology, Children′s Hospital of Chongqing Medical University, Chongqing Key Laboratory of Child Infection and Immunity, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China
  • Received:2021-03-30 Revised:2021-12-05 Published:2022-01-27
  • Contact: Bai Xiaoming; Wang Hua E-mail:XiaomingBai@CQMU.edu.cn; huawang@ hospital.cqmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China （81703124, 81801637）

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摘要： 【摘要】 目的 构建Spink5基因条件性敲除小鼠模型并鉴定其表型。方法 采用CRISPR/Cas9技术构建基因型为Mb1cre/+Spink5floxp/floxp的B淋巴细胞Spink5基因条件性敲除小鼠（敲除组），以基因型为Mb1+/+Spink5floxp/floxp的小鼠为对照组。提取小鼠脾脏单个核细胞并经流式细胞荧光技术分选B淋巴细胞及非B淋巴细胞，然后分别进行基因型鉴定及淋巴上皮Kazal型抑制物（LEKTI）蛋白表达的测定。切取小鼠皮肤行HE染色，测量表皮厚度，免疫荧光染色测定小鼠皮肤LEKTI蛋白荧光强度。组间比较采用配对t检验或两独立样本t检验。结果 基因型鉴定结果表明，成功构建稳定的B淋巴细胞Spink5基因条件性敲除小鼠模型。Western印迹显示，条件性敲除小鼠B淋巴细胞中LEKTI蛋白相对表达量为0.01 ± 0.02，显著低于非B淋巴细胞（0.66 ± 0.11，t = 9.99，P < 0.001）及对照组小鼠B淋巴细胞（1.08 ± 0.13，t = 13.78，P < 0.001）。39只敲除组小鼠中，4只出现皮肤干燥、散在鳞屑性肥厚型斑丘疹。敲除组皮损部位表皮厚度为（90.42 ± 21.31） μm，显著高于其非皮损部位［（29.71 ± 3.63） μm，t = 5.05，P = 0.002］及对照组表皮［（12.42 ± 2.21） μm，t = 6.74，P < 0.001］。免疫荧光检测显示，敲除组皮损处LEKTI蛋白荧光强度为46.21 ± 1.21，非皮损处为46.62 ± 2.13，与对照组（47.69 ± 1.71）差异均无统计学意义（P > 0.05）。结论 成功构建了B淋巴细胞Spink5基因条件性敲除小鼠模型，为进一步探究Netherton综合征皮肤屏障缺陷伴免疫功能异常的机制提供研究基础。

关键词: Netherton综合征, 小鼠, 基因敲除, B淋巴细胞, Spink5基因, LEKTI蛋白, 皮肤屏障受损

Abstract: 【Abstract】 Objective To construct a serine protease inhibitor Kazal type-5 （Spink5） conditional knockout mouse model, and to identify its phenotype. Methods B cell-specific Spink5 conditional knockout mice of genotype Mb1cre/+Spink5floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated protein 9 （Cas9） technology, and served as the knockout group. Mice of genotype Mb1+/+Spink5floxp/floxp served as the control group. The mice of genotype Mb1cre/+Spink5floxp/floxp or Mb1+/+Spink5floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor （LEKTI） was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group （0.66 ± 0.11, t = 9.99, P < 0.001）, and that in the B lymphocytes in the control group （1.08 ± 0.13, t = 13.78, P < 0.001）. Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group （29.71 ± 3.63 μm, t = 5.05, P = 0.002） and that of normal skin tissues in the control group （12.42 ± 2.21 μm, t = 6.74, P < 0.001）. Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues （46.21 ± 1.21）, non-lesional skin tissues （46.62 ± 2.13） in the knockout group and normal skin tissues in the control group （47.69 ± 1.71, P > 0.05）. Conclusion The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.

Key words: Netherton syndrome, Mice, knockout, B lymphocytes, Spink5 gene, LEKTI, Skin barrier dysfunction