The Effects Of Insulin Concentration On The Ability Of EB To Express Pluripotency And Differentiation Related Transcription Factors

To investigate the effects of different insulin concentrations on the growth and differentiation of EB cultivated by suspension method.EBs were cultivated in suspention in vitro for 7days to simulate the early course of differentiation from ICM to three germ layers.Different concentrations of insulin (0pM,6pM,60pM,600pM,6nM) was added to the cultivation environment.EBs were collected at day 3,5,7.And the expression of Oct4, Nanog, Gata6, Brachyury, Fgf5 was detected by RT-PCR.1. EBs with insulin were in better condition than EBs without insulin.2. The expression of Oct4 and Nanog was stronger in EBs with insulin than in EBs without insulin, the trend of group 0pM,6pM,60pM was descend, while the trend of group 600pM,6nM was fisrt ascend then descend.3. The Gata6 expression trend of EBs with insulin was ascend, the expression peak was in 60pM group at day7,EBs with insulin express much stronger Gata6 than EBs without insulin. 4. The expression of Brachyury in low insulin concentration groups 6pM,60pM was first detected at day 3, the peak was in 60pM group, the trend was descend, the expression of EBs in high concentration groups was delayed,600pM group was first detected at day 5, and 6nM group was first detected at day 7.5. The EBs with low insulin concentration (6pM and 60pM) express Fgf5 as early as day 3, the expression trend was first ascend then descend, the trends of 600pM,6nM group were ascend and EBs with 6nM insulin first express Fgf5 at day 5.6. ES cells express stronger Oct4 and Nanog while the expression of Gata6, Brachyury, Fgf5 couldn't be detected in ES cells.60pM Insulin concentration could promote the growth and differentiation of EBs, insulin of high concentrations (600pM,6nM) block the differentiation, no inluslin and 6nM insulin have negative effect on EB growth.