Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (20): 3660-3665.doi: 10.3969/j.issn.1673-8225.2010.20.012

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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*

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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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