The Effect Of Insulin On The Time-specific Expression Of Sox2,Fgf5,Pax6and Arx, Which Related To Neuron Development In Embryoid Body

Objective:To investigate the effects of different concentrations insulin on the time-specific expression of neural-specific genes in the early differentiation period of the suspension cultured EBs.Methods:EBs were cultivated in suspention in vitro for11days to simulate the early development of embryo. Different concentrations of insulin (60pM,6nM respectively) was added to the cultivation medium. EBs were collected at day1,3,5,7,9and11respectively. And the expression of Sox2, Fgf5, Pax6, Arx were detected by Real-time PCR.Results:1. In terms of the morphology, EBs with60pM insulin were in better condition than EBs with6nM insulin. The capsule cavity tendency appeared at day5in60pM EBs and became more obviously at day7; while there were no obvious cystic EBs in6nM group through all over11days.2.A significant upregulation of neuronal marker genes(Sox2, Fgf5, Pax6, Arx) were observed in EBs with6nM insulin, compared with that with60pM insulin.3. The expression peaks of Sox2in both groups were detected at day5,then descended to the lowest level at day11. 4. The expression peaks of Fgf5were detected at day7in60pM EBs and at day9in6nM EBs, and then descend to the same lowest level at day11in both groups.5. Pax6expression of EBs in6nM group began to rise from day3, reached the peak at day7, and descended to the lowest level at day11. However, the Pax6expression in60pM EBs was very low all over11days compare with6nM insulin group.6. Arx expression of EBs in both groups was very low in the first5days, began to rise at day7, reached the peak at day9, then descended to the same low level at day11. However, Arx expression was much stronger in6nM group than in60pM group at day7and day9.Conclusion:1. The expression of Sox2, Fgf5, Pax6, Arx show dynamic changes with different culture time.2. The expression of neural specific genes (Sox2,Fgf5,Pax6and Arx) is different in EBs which cultured with varies concentration of insulin. Compared with physiological concentrations(60pM) insulin,6nM insulin could enhance the expression of neural specific genes in EBs.