Transfection of stem cells derived from rat dental pulp with green fluorescent protein infection by lentiviral vector
10.3969/j.issn.2095-4344.2014.45.016
- VernacularTitle:慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞
- Author:
Ruihan ZHANG
;
Jia LIU
;
Shanshan NIE
;
Xuan WANG
;
Boqi LI
;
Dalei SUN
;
Dilimaolati REFUKATI
;
Yishan LIU
- Publication Type:Journal Article
- Keywords:
stem cel s;
dental pulp;
lentivirus infections;
green fluorescent proteins
- From:
Chinese Journal of Tissue Engineering Research
2014;(45):7299-7305
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Stable and efficient labeling of dental pulp stem cel s in vitro is most important in tracer technique, which is also the basis of tooth regeneration in vivo.
OBJECTIVE:To determine the optimal condition and method for transfection of stem cel s derived from rat dental pulp with green fluorescent protein infection by lentiviral vector and to determine whether green fluorescent protein-labeled dental pulp stem cel s maintain their stem cel properties.
METHODS:Rat dental pulp stem cel s were obtained by modified enzyme digestion method, to identify the immune phenotype and differentiation potential. Dental pulp stem cel s were infected with green fluorescent protein by lentiviral vector for 24 and 48 hours at different multiplicity of infection (MOI) (5, 10, 25, 50 and 100). The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy. The clonal and proliferation ability, cel cycle and the mineralization potential were compared before and after transfection. Based on those mentioned above, we could evaluate the influence of infection on their biological characteristics.
RESULTS AND CONCLUSION:Flow cytometry results showed that rat dental pulp stem cel s expressed STRO-1 and CD146 but not CD34 or CD45. The dental pulp stem cel s could differentiate into osteoblasts and
adipocytes when cultured in specific medium for each lineage differentiation. The highest efficiency of infection and strongest fluorescence expression appeared at 48 hours of infection and MOI 50. There were no significant differences in growth ability, cel colony formation rate and cel cycle before and after transfection (P>0.05). And the alkaline phosphatase expressed positively. Infection for 48 hours at MOI 50 is optimal for transfecting dental pulp stem cel s with green fluorescent protein by a lentiviral vector, thereby providing reliable tracer method for the study of rat dental pulp stem cel s in vivo.
