| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 103 === Abstract
Mesenchymal stem cells (MSCs) are multipotent adult stem cells that are able to differentiate into mesoderm layer. MSCs have exhibited the immunosuppression. Our lab has established the adult stem cells from amniotic fluid of the C57BL/6J mouse, named as AFSCs. Our previous study had discovered that cultured with activated T cells and AFSCs together contained high concentrations of nitric oxide (NO) in the supernatants, and this increase in NO is time-dependent. It has been known that NO is involved in adult bone marrow-derived MSC-mediated immunosuppression, but its role in fetal MSCs such as AFSC remained unknown. As well as, our previous RT-PCR results have been shown that AFSCs expressed several growth factors included KGF (keratinocyte growth factor). KGF has been reported involved in T-cell-mediated GvHD (graft-verse-host disease) prevention. In addition, how MSCs affected T cell receptor (TCR) signalling after stimulation has not been reported yet. Phosphorylating MAPK family proteins can initiate T cell activation, in contrast, phosphorylating hematopoietic-specific serine-threonine protein kinase (HPK1), a negative regulator of TCR signal, can turn off TCR signal activation. However, MSC’s actions on downstream stimulatory or inhibitory pathway of TCR signalling have remained to be elucidated. Therefore, there are two goals in my thesis: the first is to examine the possible molecules or soluble factors involving in AFSC-mediated T cell immunosuppression, and the second is to investigate the impact of AFSCs on both MAPK and HPK1 signals of T cell activation. Results of first part of research study indicated that the production of NO is increased in AFSC and activated T cells co-cultures. Results of RT-PCR had shown that AFSCs do not express nNOS, but eNOS was consistently present in AFSCs with or without activated T cells. In addition, iNOS expression was significantly induced in AFSCs upon its co-culture with T cells with or without anti-CD3/CD28Ab stimulation. The NO-mediated T cell proliferative suppression in AFSC culture is attenuated by added NOS inhibitor, L-NMMA. It well accepted that the transcriptional expression of iNOS is regulated by IFN- signal. AFSC co-culture is also increased IFN- production. Results of antibody block assay have showed that IFN-γ appeared to be the key player in NO-mediated T cell proliferative suppression in AFSC co-culture when anti-IFN-γ mAb was added to the co-culture for blocking. By adding anti-KGF mAb into the anti-CD3/CD28 mAbs stimulated T cell co-cultured with AFSCs, T cell proliferation was restored to a significant level, however, level of NO production has not been greatly reduce, suggesting KGF-mediated T cell proliferative suppression in AFSC is NO-independent. In terms of the second part results, AFSC co-culture is also decreased IL-2 production following with suppressed T cell proliferation. MAPK was investigated in terms of its phosphorylation levels within the T cells under different conditions. When stimulated T cells were co-cultured with AFSCs, phosphorylation level was greatly weakened in all of p38, ERK, and JNK - MAPK subfamilies, in comparison to control group of simulated T cells in AFSC absence. Correspondingly, when stimulated T cells were co-cultured with AFSCs, phosphorylation of HPK1 appeared to persist at its optimal level throughout the stimulation time course. In summary of the above results indicated that AFSCs possess a potential immunosuppressive effect on stimulated T cells by using two very different mechanisms: one being the secretion of soluble factors such as NO, IFN-, and KGF to suppress T cell proliferation, the other being a signal transduction through the negative regulator HPK1 in cell-to-cell contact.
Key Words: Mesenchymal stem cells, amniotic fluid stem cells, NO, KGF, MAPK, HPK1
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