Effects of Transfection of Keratinocyte Growth Factor Gene derived from Dendritic Epidermal T Cell on Keratinocyte Proliferation and Differentiation

Seo, Seong Jun; Ahn, Seung Won; Kim, Yong Joon; Hong, Chang Kwun

Abstract

BACKGROUND: Dendritic epidermal T cell(DETC) resident in the mouse epidermis is gammadelta-T lymphocytes. Most of them express Vgamma5/Vdelta1 T cell receptor. Compared with alphabeta-T lymphocytes, physiologic and/or pathologic role of DETC are not fully understood. Keratinocytes under the conditions such as irradiation, viral and bacterial infection, and exposure to carcinogen may express keratinocyte self-antigen. DETC may have the ability to recognize stressed and malignant keratinocytes. Moreover, specific chemokines released from DETC may contribute to inflammatory response, which maintain skin integrity and homeostasis.
OBJECTIVE
The purpose of this study was to investigate the effects of transfection of keratinocyte, growth factor(KGF) gene derived from DETC on the keratinocyte proliferation and differentiation in cell culture.
METHODS
RT-PCR using KGF primer to confirm the KGF mRNA in DETC and Keratinocyte MTT assay to evaluate the effect of keratinocyte proliferation and immunohistochemical staining using anti keratin(K)14 mAb, anti K1,10 mAb and anti involucrin mAb as a marker to elucidate the effect of keratinocyte differentiation were done.
RESULTS
1.The KGF specific PCR product was demonstrated in DETC, normal positive control of cultured fibroblast and KGF gene transfected cultured keratinocytes, but it was not observed in normal cultured keratinocytes by RT-PCR using KGF-specific primer. 2.Numbers of keratinocytes in normal control group were (2.5+/-0.5)X10(4), (8.8+/-1.5)X10(4), (8+/-0.5)X10(4), (10.5+/-0.7)X10(4) after 1st, 2nd, 3rd and 4th days, respectively. Numbers of keratinocytes with KGF gene transfected group were (1.5+/-1.0)X10(4), (9.5+/-0.2)X10(4), (9.8+/-0.5)X10(4), (13+/-2.0) X10(4) after 1st, 2nd, 3rd and 4th days, respectively. In comparison to the control, the numbers of keratinocytes were increased in the KGF gene transfected group from the 3rd and 4th day(p<0.05). On the MTT assay, compared with the control, the numbers of keratinocytes were also increased in the KGF gene transfected group at 3rd day(p<0.05). 3.In the immunohistochemical staining with mAb to K1,10 and involucrin, to evaluate the degree of differentiatin of kerationcyte reveals that KGF gene transfected cells with Ca++(0.5mM) were stained weaker than the degree of differentiation of Ca++(0.5mM) treated group. Immunohistochemical staining with mAb to K14 reveals that KGF gene transfected cells with Ca++(0.5mM) were stained stronger than the degree of differentiation of Ca++(0.5mM) treated group.
CONCLUSION
The above results strongly suggest that KGF, which produced by activated DETC, have a role in stimulating the proliferation and suppressing the differentiation of keratinocytes in the skin. In conclusion, DETC may involved in the control of proliferating activity of neighboring kerationcytes through KGF communication, which play an important role in the maintenance of epithelial integrity.