蛋白激酶C抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的初步研究

A preliminary experimental study on dedifferentiation of epidermal keratinocytes into epidermal stem cells induced by protein kinase C inhibitor GF109203X

目的探讨蛋白激酶C(PKC)抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的可能性。方法采用酶消化法结合Ⅳ型胶原快速贴壁法获得人原代表皮干细胞及角质形成细胞,倒置相差显微镜下观察细胞生长状况,免疫细胞化学染色法检测GF109203X诱导培养后角质形成细胞的表型及功能改变。同期分离培养的人在体表皮干细胞作为本次实验的阳性对照,原代角质形成细胞培养2 d后加等体积二甲基亚砜为阴性对照。结果表皮干细胞快速黏附,培养4 d后细胞呈圆形,形态规则,折光性强,明显克隆,β1整合素、CK19及CK14呈阳性表达,CK10阴性表达;已分化角质形成细胞不能快速黏附,培养4 d细胞呈类圆形,大小不一,折光性较差,无明显克隆,β1整合素、CK19及CK14呈阴性表达,CK10呈阳性表达。角质形成细胞经GF109203X诱导培养2 d后,实验组与阳性对照组细胞群β1整合素、CK19、CK14均呈阳性表达,但实验组较阳性对照组中细胞β1整合素、CK19、CK14阳性细胞数少,CK10均呈阴性表达;阴性对照组中细胞群β1整合素、CK19及CKl4呈阴性表达,CKl0呈阳性表达。结论 GF109203X能够诱导角质形成细胞去分化形成表皮干细胞。

Objective To investigate the feasibility of dedifferentiation of epidermal keratinocytes into epidermal stem cells induced by protein kinase C inhibitor GF109203 X. Methods Human primary epidermal stem cells and epidermal keratinocytes were obtained by enzyme digestion method and type Ⅳcollagen coated chosen method. The cells were divided into 3 groups as follow： experimental group： primary epidermal keratinocytes were cultured for 2 days before treated with GF109203 X, of which the final concentration was 10 μM; negative controlled group： the same volume of DMSO was given instead of GF109203 X as in experimental group; positive controlled group： human primary epidermal stem cells cultured in the same period without any intervention except for changing medium every other day. Growth of the cells cultured in vitro was observed by inverted microscope. Monoclonal antibody of integrinβ1, keratin 19（CK19）, CK14, and CK10 were detected by immunocytochemical staining. Results Epidermal stem cells exhibited rapid adherence to typeⅣ collagen, the morphology of the cells was round, which had refractivity and formed distinct clones after 4 days culture, the expressions of integrinβ1, CK19 and CK14 were positive. Epidermal keratinocytes could not adhere rapidly to typeⅣ collagen, the morphology of the cells was close round, with different size, which had no refractivity and formed few clones after 4 days culture, the expression of CK10 was positive. Immunocytochemical staining showed that the expressions of integrinβ1, CK19 and CK14 were positive in experimental group and negative in negative controlled group, but the former exhibited less positive cells after being treated with GF109203 X for 2 days, for the both of the two groups, CK10 expression was negative. Positive controlled group showed the opposite result： the expressions of integrinβ1, CK19 and CK14 were negative and CK10 was positive. Conclusion GF109203 X may induce the epidermal keratinocytes to convert into epidermal stem cells, which provide a nontransgenic approach for dedifferentiation of epidermal keratinocytes into epidermal stem cells. On the other hand, the result of the study may offer a new idea for dedifferentiation of epidermal keratinocytes into induced pluripotent stem cells（i PSC）.