胎鼠表皮干细胞的体外分离培养及rAAV_2/eGFP转染的研究

ISOLATION AND CULTURE OF FETAL MURINE EPIDERMAL STEM CELLS IN VITRO AND rAAV_2/eGFP GENETIC TRANSFECTION

目的对表皮干细胞(ESCs)进行分离培养和鉴定,利用携带绿色荧光蛋白(eGFP)基因的重组腺相关病毒(rAAV)转染表皮干细胞,探索不同滴度rAAV2/eGFP的转染效率。方法1.获取胎鼠表皮单细胞悬液。2.利用层黏连蛋白(laminin)和Ⅳ型胶原(CollagenⅣ)筛选ESCs,在体外进行培养和鉴定。3.将ESCs按5×104个/孔接种入24孔培养板中,按照不同的病毒基因数与转染细胞数之比(MOI)加入所需rAAV2/eGFP病毒,计数绿色荧光细胞数目,并计算转染效率。结果1.ESCs对Laminin和CollagenⅣ的吸附性很好,克隆形成率较高。2.对ESCs进行β1整合素(Integrinβ1)单抗、角蛋白19(Keration 19,K19)单抗的免疫细胞化学染色,均呈阳性表达。3.利用rAAV/eGFP病毒体外转染ESCs,可稳定表达eGFP。结论用Laminin和CollagenⅣ快速黏附法可从胎鼠皮肤分离和富集ESCs。

Objective To isolate, culture and identify epidermal stem cells （ESCs）. The gene of enhanced green fluorescent protein（eGFP） was introduced via recombinant adeno-associated virus（rAAV） infection into the epidermal stem cells in vitro and the transfection efficiency under various tite of culture was determined. Methods 1. Getting fetal Wistar rats＇ dissociated single epidermal cells. 2. Making laminin and Collagen IV the substitution of basal memrane, ESCs were isolated by adhering to murine laminin and Collageniv .3. Epidermal stem cells were cultured in twenty-four-well plate ,and cells number was controled 5 × 10^4 or so. After epidermal stem cells adhered to plate, diluted rAAV2/eGFP virus fluid with serum-free medium. The diluted rAAV2/eGFP virus fluid was added to the twenty-four-well plate according to the different MOI（viru gene/cells）. The number of green fluorescence cells were counted under fluorescence microscope. The transfection efficiency was determined. Results 1. ESCs had better adhesive ability to laminin and higher colony formation efficiency（CFE） than that of keratinocytes. 2. ESCs wer strongly positive with immunocytochemical staining of integrin β1 and keration 19（K19）.3. After rAAV2/eGFP genetic transfection of ESCs, the positive cloning expressed highly eGFP gene along with time. Conclusion 1. These results suggest that rats＇epidermal stem cells could been successfully isolated and enriched in vitro by means of rapid adherence to laminin and Collagen Ⅳ . After rAAV2/eGFP genetic transfection of epidermal stem cells, the positive cloning expressed highly eGFP gene stably along with time.