OBJECTIVE： To investigate the effects of resveratrol （RV） in reprogramming mouse embryonic fibroblasts （MEFs） into induced pluripotent stem cells （iPSCs） and the related mechanism. METHODS： Primary MEFs were isolated from E13.5 embryos and used within three passages. Retroviruses expressing Sox2 and Oct4 were produced by transfecting GP2-293t cells with recombinant plasmids murine stern cell virus （MSCV）-Sox2 and MSCV-Oct4. Supernatants containing retroviruses were obtained after 48-hour transfection and MEFs were then infected. Different concentrations （0, 5, 10 and 20 IJmol/L） of RV were added to embryonic stem cell （ESC） medium to culture MEFs 48 h post-infection, iPSC clones emerged and were further cultured. Expression of pluripotent markers of iPSCs was identified by cell immunofluorescence and reverse transcription-polymerase chain reaction. Both cytotoxicity and cell proliferation were assayed by Western blot analysis after RV was added into ESC medium. The ultrastructure change of mitochondria was observed by electron microscopy. RESULTS： More than 2.9-fold and 1.3-fold increases in colony number were observed by treatment with RV at 5 and 10 pmol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 pmol/L RV. The proliferation effect on MEFs or MEFs infected by two factors Sox2/Oct4 （2 factors-MEFs, 2F-MEFs） was investigated after RV treatment. At 20 pmol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 IJmol/L treatments. Clones were selected from the 10 pmol/L RV-treated group and cultured. Green fluorescent protein expression from one typical clone was silenced one month later which expressed ESC-associated marker genes Gdf3, Nanog, Ecatl, Fgf4 and Foxd3. Electron transmission microscope showed obvious cavitations in mitochondria. The expression of hypoxia-inducible factor-la was up-regulated when 2F-MEFs were treated with RV compared to the control group. CONCLUSION： RV imp
Dao-fang DingXiao-feng LiHao XuZhen WangQian-qian LiangChen-guang LiYong-jun Wang
