ISOLATION AND CULTURE OF FETAL MURINE EPIDERMAL STEM CELLS IN VITRO AND rAAV_2/eGFP GENETIC TRANSFECTION
- VernacularTitle:胎鼠表皮干细胞的体外分离培养及rAAV_2/eGFP转染的研究
- Author:
Ximei ZHANG
;
Yunqiu GUO
;
Lianhong JIN
- Publication Type:Journal Article
- Keywords:
Skin;
Stem cells;
Immunocytochemistry;
Genetic transfection;
Rat
- From:
Acta Anatomica Sinica
2002;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
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 CollagenⅣ 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 rAAV_2/eGFP virus fluid with serum-free medium.The diluted rAAV_2/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 ?1 and keration 19(K19).3.After rAAV_2/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 CollagenⅣ.After rAAV_2/eGFP genetic transfection of epidermal stem cells,the positive cloning expressed highly eGFP gene stably along with time.
