| Protein-specific expression has become a hot research spot recently. Many studies have already proved the regulatory effect of activation and inactivation of proteins in both physiological and pathological development. Tissue factor (TF) is a transmembrane single-chain glycoprotein, composed of263amino acid residues. It plays an important role in maintaining blood coagulation balance and protecting vital organs by activation and specific expression in physiological and pathological conditions. Meanwhile, the expression of TF in hematopoietic cells also shows significant differences. However, the regulatory mechanism remains unknown. Epigenetics, as an important branch of biological genetics, has become a focal point of life science nowadays. It is accepted that the epigenetic regulation of gene affects the expression of protein and the differentiation of various types of cells. At the same time, human embryonic stem cells (hESCs) has become an important experimental object in the field of regenerative medicine due to their multi-differentiation potential and the character of self-rooted maintain, which allows them to imitate the process of tissue and organ development.In conclusion, in this experiment, we successfully established a system for specific differentiation from embryonic stem cells to hematopoietic stem cells and trophoblast cells. We tested the expression of TF in different stages of differentiation and different cell types. And then, discussed if the results was related to epigenetic regulation. Based on the above purpose, this study is divided into the following three parts.Chapter1Here we induced the differentiation of HSC cells by co-culture of embryonic stem cells (hESCs) and mouse bone marrow stromal cells (OP9). Decrease expression of stem cell specific markers can be detected by RT-PCR and flow cytometry with the induction of differentiation, which is companied with an increase expression of hematopoietic related specific markers. CD34+cells are purified by MACS system, and then induced to myeloid, erythroid and megakaryocyte by different combinations of cytokines in vitro. Increasing expression of differentiation related genes can be detected by RT-PCR and flow cytometry. We used Marixgel and BMP4to induce the differentiation of trophoblast cells, of which the expression of cell-specific marker can be detected by RT-PCR, immunofluorescence technique and flow cytometry. Conclusion:An effective platform to induce human embryonic stem cells differentiation has been established, which provides a reliable source of cell samples for further research.Chapter2We detected the expression changes of tissue factor in the induction process from human embryonic stem cells to hematopoietic stem cells, blood cells and trophoblast cells by RT-PCR, TF activity assay and flow cytometry. In the process of differentiation from human embryonic stem cell to hematopoietic stem cell, TF expression was hardly detected by RT-PCR, flow cytometry and TF activity assay. In the process of differentiation from hematopoietic stem cell to blood cell, TF expression can be detected by different methods. RT-PCR can detect TF expression in CD15+cells and CD41+cells, but doesn’t work in CD235a+cells. Flow cytometry shows the percentage of CD142expressing cells in CD15+cells, CD41+cells and CD235a+cells separately:5.83%,3.94%,1.58%. TF activity assay indicates a weak TF activity in CD15+cells only. In the process of differentiation from human embryonic stem cells to trophoblast cells, increasing expression of TF can be detected by Western blot assay, while it also can be detected by both mRNA and activity assay. Conclusion:Differences in TF expression can be detected during the development of embryonic stem cells.Chapter3The expression of TF can be regulated by methylation of DNA promoter region. We test this expression change in the process of differentiation from human embryonic stem cells to hematopoietic stem cells, blood cells and trophoblast cells by methylation specific PCR (MSP). In the process of differentiation from human embryonic stem cells to hematopoietic stem cells, the DNA promoter region of the TF gene shows a semi-methylated semi-unmethylated expression, which however is dominated by methylated expression. In the differentiation of hematopoietic stem cells, the methylation level reduces in myeloid cells compared with previous, but remains equal in other cells. In the process of differentiation from human embryonic stem cells to trophoblast cells, the DNA promoter region of the TF gene presents a semi-methylated semi-unmethylated expression. And with the formation of trophoblast cells, the methylation level decreased while the non-methylation level increased. The results indicate that DNA methylation regulates TF gene expression. |
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