中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (20): 3660-3665.doi: 10.3969/j.issn.1673-8225.2010.20.012

• 皮肤粘膜组织构建 skin and mucosal tissue construction • 上一篇    下一篇

人日光性角化病角质形成细胞的体外培养

龙庭凤1，2，何 黎1，刘 流1，沈丽达2，李坤杰1，涂 颖1   

  1. 1昆明医学院第一附属医院皮肤及医疗美容科，云南省皮肤病研究所，云南省昆明市650032；
    2昆明医学院第三附属医院，云南省肿瘤研究所肿瘤化疗研究中心，云南省昆明市  650118
  • 出版日期:2010-05-14 发布日期:2010-05-14
  • 通讯作者: 刘 流，男，博士，教授，博士生导师，昆明医学院第一附属医院皮肤及医疗美容科，云南省皮肤病研究所，云南省昆明市 650032 liuliu3939@126.com 何 黎，女，博士，教授，博士生导师。昆明医学院第一附属医院皮肤及医疗美容科，云南省皮肤病研究所，云南省昆明市 650032 heli2662@yahoo.com.cn
  • 作者简介:龙庭凤，女，1977年生，云南省永仁县人，昆明医学院在读博士，彝族，医师，主要从事皮肤肿瘤的诊断与治疗。 ltf7712@163.com
  • 基金资助:

    国家自然科学基金资助(30860257)。

In vitro culture of keratinocytes isolated from actinic keratosis

Long Ting-feng1,2, He Li1, Liu Liu1, Shen Li-da2, Li Kun-jie1, Tu Ying1    

  1. 1Department of Dermatology, the First Affiliated Hospital of Kunming Medical College, Research Center of Dermatology of Yunnan Province, Kunming  650032, Yunnan Province, China;
    2Research Center of Medical Oncology Chemotherapy, the Third Affiliated Hospital of Kunming Medical College, Kunming  650118, Yunnan Province, China
  • Online:2010-05-14 Published:2010-05-14
  • Contact: Liu Liu, Doctor, Professor, Doctoral supervisor, Department of Dermatology, the First Affiliated Hospital of Kunming Medical College, Research Center of Dermatology of Yunnan Province, Kunming 650032, Yunnan Province, China; Research Center of Medical Oncology Chemotherapy, the Third Affiliated Hospital of Kunming Medical College, Kunming 650118, Yunnan Province, China liuliu3939@126.com He Li, Doctor, Professor, Doctoral supervisor, Department of Dermatology, the First Affiliated Hospital of Kunming Medical College, Research Center of Dermatology of Yunnan Province, Kunming 650032, Yunnan Province, China heli2662@yahoo.com.cn
  • About author:Long Ting-feng, Studying for doctorate, Physician, Department of Dermatology, the First Affiliated Hospital of Kunming Medical College, Research Center of Dermatology of Yunnan Province, Kunming 650032, Yunnan Province, China; Research Center of Medical Oncology Chemotherapy, the Third Affiliated Hospital of Kunming Medical College, Kunming 650118, Yunnan Province, China ltf7712@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30860257*

PDF

378

被引次数

8

摘要：

背景：国内外相关研究已建立和发展了多种疾病状态下角质形成细胞的培养方法，但目前关于人日光性角化病角质形成细胞体外培养的研究未见报道。
目的：体外分离培养人日光性角化病角质形成细胞并鉴定其生物学特性。
方法：皮肤标本由昆明医学院第一附属医院皮肤科和整形外科提供。两步消化法体外分离培养日光性角化病组和正常老年人面部皮肤对照组的角质形成细胞，无血清培养技术进行人体角质形成细胞的培养，待细胞生长至70%~80%融合时消化传代。在倒置相差显微镜和激光共聚焦显微镜下观察细胞形态及超微结构，应用免疫荧光检测角蛋白在细胞内的表达，划痕实验和双层软琼脂集落形成率实验观察细胞的迁移和成瘤能力。
结果与结论：原代培养的细胞显示典型的表皮细胞特征：倒置显微镜下培养的细胞呈典型的“铺路石”状，经免疫荧光检测为角蛋白阳性，电镜下可见细胞内有大量的张力纤维。体外成功分离培养出老年人病变条件下的角质形成细胞。日光性角化病组与对照组相比：细胞的核仁增大，异染色质及线粒体数量增多，内质网数量少特点，细胞较为幼稚。生长曲线、划痕实验和双层软琼脂集落形成率实验表明，日光性角化病患者的角质形成细胞较同龄老年人具有更强的增殖、迁移和成瘤的能力。实验证实体外培养的日光性角化病角质形成细胞仍具有一定的非典型增生细胞的特性。

关键词: 日光性角化病, 角质形成细胞, 分离, 培养, 生物学特性, 组织构建

Abstract:

BACKGROUND: A variety of culture methods about keratinocyte under diseases has been established and developed. However, few studies reported in vitro culture of keratinocytes isolated from actinic keratosis.

OBJECTIVE: To culture keratinocytes isolated from actinic keratosis and to identify its biological characteristic in vitro.

METHODS: The skin samples were obtained from patients who were admitted in the Department of Dermatology and Plastic Surgery of the First Affiliated Hospital of Kunming Medical College. Primary keratinocytes were isolated from patients with actinic keratosis and postoperatively skin of old people using two-step digestion technique. Serum-free culture technology was used to culture human keratinocytes. The cells were passaged when reached 70%-80% confluency. Morphological changes and ultrastructure of cells were observed under the microscope and transmission electron microscope. The expression of keratin in keratinocytes were detected by indirect immunofluorescence. In addition, the capacity of migration and tumorigenicity was observed by scratch test and double-soft-agar colony formation rate experiments.

RESULTS AND CONCLUSION: The keratinocytes possessed typical epidermal cell characteristics: that is, revealed a typical “paving stone” like under an inverted microscope, showed keratin-positive, and massive bundles of tonofibrilla could be seen in the plasma under a transmission electron microscope. Compared with the control group, nucleolus, heterochromatin and number of mitochondria were increased, number of endoplasmic reticulum was reduced, and the characters of keratinocyte under electron microscope showed more immature in the keratinocytes from actinic keratosis. The growth curve, scratch test, double-soft-agar colony formation rate experiments showed that solar keratosis keratinocytes, compared with elder age, had a stronger proliferation, migration and tumorigenic capacity. All results demonstrated that keratinocyte can keep its origin morphological characteristics during in vitro culture. 

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