| After people finding that somatic cell could fuse with virus, research of cell fusion has been deepened increasingly. Now fusion induced by virus or chemicals is used to study gene expression, mapping of genome, producing of antibody and curing of cancer. Cell fusion is important because it is contained almost in whole process of life, including development, disease occurrence and healing. It also has deeply effected on regeneration of tissues which is regulated by stem cells. One candidate mechanism of effect of stem cells is cell fusion, it is complicated but important.Many tissues can regenerate after injury, such as liver, muscle and bone. Connecting with signal of injury, stem cells in and out of the injured tissue move to the proper location and differentiate to tissue specific cells. However, this explanation of regeneration can not describe how the stem cells move to the "proper location". But if stem cells fuse with parenchymatous cells of the tissue, the doubt will be readily solved. Cell fusion has been proved to be one mechanism of tissue regeneration. Hybrid cells can proliferate and divide normally. Modified hybrid cells will be one new cell source that can be used for regenerative medicine and cell therapy. However, little was know about the function and the fate of hybrid cells.By fusing somatic cells with embryonic derived cells or by transducting several key factors, somatic cells could be reprogrammed. It is well known that reprogramming is the most likely mechanism of function of hybrid cells. After fusing with stem cells, somatic cells will be reprogrammed, in this case, the potential and fate of differentiated cells will be regulated by fusion. After formation of heter or syeh, chromosomes from both parental cells mix together, somatic cells would be reprogrammed. This also can be induced by several key factors derived from stem cells. Therefore, formation of sye will change capability and fate of somatic cells. The consequent hybrid cells are pluripotent, just the same with ES cells. So, based on these theories, this research supposed that in condition of damage, reprogrammed hybrid cells can be functional the same way like pluripotent stem cells.First of all, cell damaged model was conducted in vitro. Human embryonic hepatocytes were treated with oxidant agent H2O2. If the injury is too serious, most of the treated cells will die while mild treatment will not be sufficient for the following experiment. Therefore, cells were treated in different times and with different doses in order to select the proper condition. By analyzing cell activity, secretion of damage related enzymes and expression of functional proteins, treating effect curves were made to evaluate degree of damage. Results showed that C3=200μM with2h was the optimized condition.It has been debated for a long time that whether rare ratio of fusion was efficient for regeneration. Till now, detection of fusion ratio based on technologies using specific markers, such as FISH and existence of Y chromosome. However, all these technologies can cause high "losing rate" of chromosome. Therefore, after coculturing ES cells with GOF-18transfected injured cells, fusion ratio was detected by karyotyping directly. The results showed that proportion of fusion was at least twelve times that of ratio of non-cocultured control group, after48h’s coculture. By karyotyping and detection of fluorescence, it was indicated that the non-diploid cells were fusion production of parental cells. After detecting damage related enzymes, damage of cells was inhibited. Meanwhile, results of expression of proteins and gene expression profile showed that ES cells differentiated to hepatocytes like cells.Taking together, after coculturing ES cells and hepatic cells, fusion of parental cells occurred and damage was repaired. The ratio of fusion was far higher than ratios reported before. This result suggested that fusion did have effect on injured cells. It is interesting and important that after coculture, ES cells differentiated directly to hepatic epithelial cells. This result indicated one new method of differentiation of ES cells in vitro. Meanwhile, it also implied that whether differentiating to tissue specific cells will be one effect way of regprogrammed hybrid cells on tissue regeneration, just as the adult stem cells, because after fusing with ES cells, hybrid cells will be reprogrammed.It was proved that coculture has effect on reparation and hybrid cells have effect on repair of cell damage. Based on these, in order to study how hybrid cells effect on injured cells, we further our research to next step. By using PEG, hybrid cells derived from ES cells and GOF-18transfected cells were harvest in vitro. After that, hybrid cells were identified by several methods.85%of the selected hybrid cells were EGFP positive cells, which indicated occurring of fusion.Hybrid cells play an important role in organogenesis, tissue regeneration and cancer formation. However, the fate of hybrid cells and their capacity are poorly understood. Therefore, based on results above, the aim of this study was to elucidate the fate and function of hybrid cells in condition of injury in vitro. Numerous studies reported that hybrid cells derived from pluripotent cells and somatic cells, genome of somatic cells can be reprogrammed. Thus, we hypothesized that in environment of damage, reprogrammed hybrid cells could effect like pluripotent ES cells. Using transwell, reprogrammed hybrid cells derived from mouse ES cells and human liver cells were co-cultured with injured human hepatic cells. After co-culturing, the H2O2-induced damage in hepatic cells was ameliorated by inhibiting apoptosis. Apoptosis status was evaluated by Annexin-V/PI double-staining, FACS and Western-blot. The results showed that in the p53related signal pathway, endogenous Bax changed significantly and is one key factor that regulates inhibition of apoptosis. Furthermore, by analyzing the gene expression profile and protein expression, the fate of hybrid cells was studied. The results of RT-PCR indicated that during coculturing, hybrid cells were differentiated into hepatic epithelial cells. Hybrid cells transcripted genes from both parental cells’ genomes. By immunocytochemistry, hepatic directional differentiation of hybrid cells was confirmed. In addition, by measuring injury related enzymes and ALB release, repair of injury was studied. Compared to the control group, activity of these enzymes trended toward the level of normal hepatic cells. Stably increased levels of secretion of ALB demonstrated that differentiated hybrid cells were functional hepatic cells.After establishing of damaged cell model, we analyzed ratio of fusion and function of fusion in coculture system of ES cells and damaged cells. Based on proving that fusion did have effect on reparation of damage, we evaluated the fate and function of hybrid cells. The results showed that, in environment of damage, hybrid cells differentiated to functional hepatic cells, inhibited apoptosis and damaged cells and improved the proliferation of damaged cells.The present research studied the fate and function of hybrid cells in environment of damage for the first time in vitro. And we applied hybrid cells for researching of regeneration for the first time. Meanwhile we built up a new way to differentiate ES cells directly to hepatic cells. This research expands the knowledge of fusion-related events. Moreover, it could indicate a new route of differentiation from pluripotent cells to tissue-specific cells. |
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