| Chapter one:Neurons and astrocytes differentiated from mouse embryonic stem cells induced by IsobavachinObjective:To evaluate the inductive effects of isobavachin (IBA) on the directed differentiation of mouse embryonic stem(ES) cells into neuronal cells in vitro and study the mechanism. Methods:The hanging drop method was employed for embryonic body formation to mimic embryo development in vivo. The final concentration of IBA is 10-7mol/L,10-7mol/L RA as the positive control and 0.1% DMSO as the solvent control. At the final of differentiation, morphological evaluation and immunocytochemistry staining were conduced to indentify the neurocyte derivatives. Samples of ES, EB, d 8+0, d 8+5, d 8+10 were collected. Western blot was performed to investigate the expression of the neurocyte-specific proteins including NEFM,β-tubulinⅢand GFAP, which were regarded as the inducing effect indexes of IBA. RT-PCR was performed to investigate the expression of neurocyte-specific genes concluding OCT 3/4, nestin,β-tubulinⅢand GFAP. Moreover, Western blot was performed to investigate protein expression of p38/ERK/JNK MAPK, in order to study the mechanism of the neuronal differentiation. At the period of suspension, added GGTI-298 accompanying with IBA, to investigate the neuron fate differentiated by IBA, to confirm whether the protein prenynation is the function part of the neuronal differentiation effect of IBA. Results: The neurocyte-specific proteins were expressed in the differentiation promoted by IBA. Classic neurons were observed through morphological evaluation. The axons are three or more times longer than the cell bodies. As the results of immunocytochemistry staining, OCT 3/4 and nestin were positively expressed in the period of EBs. In the terminal point of differentiation, the neuron expressed accompanying withβ-tubulinⅢand ChAT. The GFAP were not co-expressed withβ-tubulinⅢ. As is showed in the results of Western blot, theβ-tubulinⅢand GFAP were up-regulated in a time dependent way, especiallyβ-tubulinⅢ. In the MAPK pathway, P-p38 and P-JNK was down-regulated and the p-ERK was up-regulated during the neuronal differentiation. The addition of GGTI-298 can significantly reduce the neurons induced by IBA; Conclusion:IBA can facilitate mouse ES cells differentiating into neuronal cells. The mechanism was in part involved in protein prenylation and subsequently via phos-ERK activation and phos-p38 off pathway.Chapter two:Research on neurons derived from mouse ES cells expressing JP-3,4 induced by IsobavachinJunctophilin (JP) subtypes JP-3 and JP-4 are expressed specifically in brain. They are members of membrane protein family and exclusively present in excitable cells. In general, JPs consist of a C-terminal domain spanning the membrane of endoplasmatic reticulum (ER), which serves as an intracellular calcium (Ca2+) store in excitable cells, and as a long cytoplasmatic domain interacting with plasma membrane. JPs take part in enabling communication between plasma membrane of an excitable cell and intracellular ion channels. Here we also observed whether the neuronal cells differentiated from mouse ES cells are with excitable elements. Objective:To evaluate the inductive effects of isobavachin (IBA) on the directed differentiation of mouse embryonic stem (ES) cells into exciting neuron markers of Junctophilin 3,4. Methods:The hanging drop method was employed for embryonic body formation to mimic embryo development in vivo. The final concentration of IBA is 10-7mol/L,10-7mol/L RA as the positive control and 0.1% DMSO as the solvent control. At the periods of EBs (d 4 and d 8+0), immunocytochemistry staining was conducted to evaluate the neuronal progenitors of nestin and the markers of excitable neurons of JP-3, JP-4. At the final of differentiation, morphological evaluation and immunocytochemistry staining were conduced to indentify the neurocyte derivatives and the excitable neuron markers. Samples of ES, EB, d 8+0, d 8+5, d 8+10 were collected. Western blot was performed to investigate the expression of the neurocyte-specific proteins includingβ-tubulinⅢ, JP-3 and JP-4. Results:The neurocyte-specific proteins were expressed in the differentiation promoted by IBA. JP-3 and JP-4 were highly expressed in the period of EBs combined with the expression of nestin and were relatively reduced during the neuronal differentiation. At the terminal of neuronal differentiation, JP-3 and JP-4 were rare and were mainly expressed in the neuron cells. Conclusion:IBA can facilitate mouse ES cells differentiating into neurons, which express exciting neuron markers of JP-3 and JP-4. |
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