| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 97 === Mesenchymal Stem Cells (MSCs) are the adherent, spindle- or fibroblast shape cells of capable differentiating into multiple lineages of the mesoderm. Accumulated evidences indicated that MSCs exhibit immunosuppressive effect on allogenic T cells, B cells, natural killer cells (NK cells), and dendritic cells (DC) of proliferation and activation. In previous research, a primary cell line from amniotic fluid of 13-day gestation C57BL/6-GFP mice was established and named as amniotic fluid stromal cells-1 (AFSC-1). After characterized, AFSC-1 exhibited the properties of stem cells without caused tumorgenesis. Moreover, AFSC-1 inhibited Con A or anti-CD3 Abs stimulated allogenic T cell proliferations in MLR culture assay. Results of transwell culture assay indicated soluble factors were involved in AFSC-1-mediated immunosuppression. Related studies have demonstrated that soluble factors such as IL-10, IL-6, TGF-β1, IFN-γ, and NO may play the important role in the mechanisms of MSCs-mediated T cells proliferative suppression. Beside, a 70-kDa cell-surface protein with immunosuppressive activity is first identified in peripheral cytotrophoblasts of early placentas and named as regeneration and tolerance factor (RTF). RTF is a subunit of 70k-Da vacuolar ATPase (v-ATPase). RTF have been described in a broad of spectrum of tissues in human (hRTF) and mice (mRTF). RTF is cleaved to yield a membrane-bound 50-kDa protein and a secreted, biologically active 20-kDa fragment (soluble RTF), the latter can up-regulates the production of IL-10. It had been reported that mRTF inhibits allogeneic T cells proliferation, hence, mRTF have been presumably possessed the capability of immunosuppression. Therefore, This study aims at evaluating possible soluble factors on AFSC-1 inhibited T cells proliferation and the possible role of mRTF involved in AFSC-1 mediated T cells proliferative suppression.
The results are showed that the expressed levels of IL-10, TGF-β1 and IFN-γ are decreased, but the IL-6 production was significantly increased after AFSC-1 co-cultured with allogeneic T cells, however, the AFSC-1-mediated T cell proliferative suppression did not attenuated while a IL-6 neutralized antibodies were added into the culture. Therefore, IL-6 dosen't play a role in AFSC-1 inhibited T cell proliferation. Furthermore, the expressed levels of nitric oxide (NO) were increased obviously in the supernatant of AFSC-1 co-culture with allogeneic T cells. Hence, NO may be involved in AFSC-1 mediated-immunosuppression of allogenic T cell proliferation and required further study. In addition, results of RTF studies indicated that thymocytes, unactivated CD3+ T cells, Con A activated T cells (T/Con A), and CD4＋CD25＋FoxP3＋ Treg cells (Treg) were all expressed RTF factor. The expression of mRTF was decreased in AFSC-1 after co-culture with activated with Con A-stimuualted T cells, but increased in Con A-activated T cells. The expression of mRTF were also increased after AFSC-1 stimulated by IFN-γ, but it decreased after 48hrs. However, the AFSC-1 mediated Con A-activated T cell proliferative suppression was attenuated in co-culture of Con A activated T cells with AFSC-1 after pretreated with Concanamycin A (CMA), a specific v-ATPase inhibitor. This result illustrated mRTF in AFSC-1 may involve in AFSC-1 inhibited T cells proliferation. In this study, we also produce an anti-50k Da-mRTF polycolonal rabbit antibody. This antibody can detect the expression of mRTF factor in AFSC-1. In summary, NO and RTF may involve in AFSC-1 mediated T cell suppression. In the future, a RTF-specific RNA interference and iNOS inhibitor will be considered to examine the role of NO and RTF in AFSC-1-mediated immune suppression.
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