中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (6): 501-507.doi: 10.35541/cjd.20210874

• 论著 • 上一篇    下一篇

人乳头状瘤病毒16 E6/E7基因稳定表达的永生化角质形成细胞的建立及鉴定

许翠1    何勇2    吴亦琳2    吕群2    李黎明2    蒋明军2   

  1. 1南京大学医学院附属鼓楼医院皮肤性病科，南京  210008；2中国医学科学院、北京协和医学院皮肤病研究所中心实验室，南京  210042 
    许翠和何勇对本文有同等贡献 

  • 收稿日期:2021-12-02 修回日期:2022-04-30 发布日期:2022-06-02
  • 通讯作者: 李黎明; 蒋明军 E-mail:llming@pumcderm.cams.cn; drmingjunjiang@ 163.com
  • 基金资助:
    江苏省自然科学基金（BK20191136）；江苏省重大疾病生物资源样本库开放课题（SBK202005003）；北京协和医学院“中央高校基本科研业务费”项目（3332019104）；南京市卫生科技发展专项资金项目（YKK20074）

Establishment and identification of human immortalized keratinocytes stably expressing human papillomavirus type 16 E6/E7 gene

Xu Cui1, He Yong2, Wu Yilin2, Lyu Qun2, Li Liming2, Jiang Mingjun2   

  1. 1Department of Dermatology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China; 2Central Research Laboratory, Institute of Dermatology, Chinese Academy of Medicine Sciences and Peking Union Medical College, Nanjing 210042, China
    Xu Cui and He Yong contributed equally to the article
  • Received:2021-12-02 Revised:2022-04-30 Published:2022-06-02
  • Contact: Li Liming; Jiang Mingjun E-mail:llming@pumcderm.cams.cn; drmingjunjiang@ 163.com
  • Supported by:
    Natural Science Foundation of Jiangsu Province of China （BK20191136）; Open Project of Jiangsu Biobank of Clinical Resources （SBK202005003）; Fundamental Research Funds for the Central Universities （3332019104）; Nanjing Health Science and Technology Development Special Fund（YKK20074）

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摘要： 【摘要】 目的 构建人乳头状瘤病毒（HPV）16 E6/E7基因稳定表达的人永生化角质形成细胞（KC），为研究HPV16 E6/E7诱导的细胞永生化及恶性转化机制提供细胞模型。方法 两步消化法分离培养原代人包皮角质形成细胞（HFK），利用慢病毒感染技术对细胞稳定转染HPV16 E6/E7基因，连续培养30代以上，筛选出永生化KC，分为3组：①空白对照组：传代2次的原代HFK；②实验组：传代2次的原代HFK感染LV5-HPV16 E6/E7，感染细胞记录为A0代，感染后以传代次数记录（A1、A2……）；③阳性对照组：HPV16阳性宫颈癌细胞SiHa。应用实时荧光定量PCR（qRT-PCR）、Western印迹实验分别检测空白对照组、实验组、阳性对照组HPV16 E6/E7 mRNA、蛋白及CK14蛋白的表达，CCK-8及Transwell Insert方法检测细胞的增殖及侵袭能力。裸鼠致瘤实验检测实验组A30、阳性对照组SiHa细胞的致瘤能力。结果 成功分离原代HFK。LV5-HPV16 E6/E7重组质粒感染原代HFK后，空白对照组细胞无荧光表达，连续传代后出现衰老表现，实验组A30细胞体积、形态较原代HFK无明显变化，且荧光表达率为100%。与空白对照组相比，实验组A1、A10、A20、A30细胞HPV16 E6 mRNA表达水平显著升高（t值分别为7.12、8.07、6.53、5.66；P值分别 < 0.001、 < 0.001、 = 0.001、 = 0.005）；与空白对照组相比，实验组A1、A10、A20、A30细胞HPV16 E7 mRNA表达水平也显著升高（t值分别为3.20、4.29、3.75、4.22；P值分别为0.024、0.008、0.013、0.014）。空白对照组未见HPV16 E6/E7蛋白的表达，而A30及SiHa细胞可见HPV16 E6/E7蛋白的表达。CCK-8实验显示，实验组A10、A20、A30细胞增殖水平显著高于空白对照组（t值分别为6.49、7.55、9.43；P值分别为0.003、0.002、0.001），而A1的增殖水平与空白对照组比较，差异无统计学意义（t = 2.40，P = 0.074）。Transwell Insert侵袭实验显示，A30不能穿过基底膜，SiHa细胞可穿过基底膜被染成蓝色。裸鼠接种A30细胞2个月后无肉眼可见肿瘤，组织学亦显示无肿瘤形成，裸鼠接种SiHa细胞后可于皮下形成肿瘤。结论 通过采用慢病毒技术转染HPV16 E6/E7基因成功建立人永生化角质形成细胞，可作为HPV相关研究中的理想细胞模型。

关键词: 人乳头瘤病毒16, 角蛋白细胞, 慢病毒感染, 细胞系, 转化, HPV16 E6/E7

Abstract: 【Abstract】 Objective To construct human immortalized keratinocytes stably expressing human papillomavirus type 16 （HPV16） E6/E7 gene, and provide a cell model for studying mechanisms underlying HPV16 E6/E7-induced cell immortalization and malignant transformation. Methods Primary human foreskin keratinocytes （HFKs） were isolated by sequential two-step enzymatic digestion. Cultured HFKs were stably transfected with a HPV16 E6/E7 gene-overexpressing lentiviral vector LV5-HPV16 E6/E7, and consecutively cultured for more than 30 passages. Then, immortalized keratinocytes were screened out and divided into 3 groups: （1） blank control group: second-passage primary HFKs; （2） experimental group: HFKs transfected with LV5-HPV16 E6/E7 at different passages, and the second-passage primary HFKs transfected with LV5-HPV16 E6/E7 were referred to as A0 cells, thereafter, the transfected HFKs were named according to their passage number, such as A1, A2, ... A30; （3） positive control group: the HPV16-positive cervical cancer cell line SiHa. Real-time fluorescence-based quantitative PCR （qRT-PCR） and Western blot analysis were performed to determine the mRNA expression of HPV16 E6/E7 and protein expression of HPV16 E6/E7 and CK14, respectively, in the blank control group, experimental group and positive control group. Cell counting kit-8 （CCK8） assay and Transwell insert invasion assay were conducted to assess the cellular proliferative and invasive activity. In vivo tumor formation experiment in nude mice was conducted to investigate the tumorigenicity of A30 cells in the experimental group and SiHa cells in the positive control group. Results Primary HFKs were successfully isolated. After the primary HFKs were transfected with the recombinant plasmid LV5-HPV16 E6/E7, the blank control group showed no fluorescence in the cells, but showed senescent phenotypes after serial passages, while in the experimental group, the volume and morphology of A30 cells were similar to those of the primary HFKs with the proportion of fluorescence-positive cells being 100%. Compared with the blank control group, the experimental group showed significantly increased mRNA expression levels of HPV16 E6 and E7 in A1, A10, A20 and A30 cells （HPV16 E6: t = 7.12, 8.07, 6.53, 5.66, P < 0.001, < 0.001, = 0.001, = 0.005, respectively; HPV16 E7: t = 3.20, 4.29, 3.75, 4.22; P = 0.024, 0.008, 0.013, 0.014, respectively）. The protein expression of HPV16 E6/E7 was absent in the blank control group, but was observed in A30 and SiHa cells. CCK8 assay showed that the proliferative activity of A10, A20 and A30 cells was significantly higher than that of the blank control group （t = 6.49, 7.55, 9.43, P = 0.003, 0.002, 0.001, respectively）, while there was no significant difference in the proliferative activity between A1 cells and the blank control group（t = 2.40, P = 0.074）. Transwell insert invasion assay showed that A30 cells could not cross the basement membrane, but SiHa cells could pass through the basement membrane and were stained blue. Two months after the inoculation with A30 cells in the nude mice, no visible tumors were found, which was confirmed by a histological study. Subcutaneous tumors were formed in the nude mice after the inoculation with SiHa cells. Conclusion Human immortalized keratinocytes were successfully established by lentivirus-mediated transfection with HPV16 E6/E7 gene, and can serve as an ideal cell model for HPV-related research.

Key words: Human papillomavirus 16, Keratinocytes, Lentivirus infections, Cell line, transformed, HPV16 E6/E7