| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 99 === Mesenchymal stem cells (MSCs) have been used in clinical trials for different disease models. Bone marrow is the traditional source of human MSC, but human term placenta appears to be an alternative and more readily available source. Previously, placenta derived mesenchymal stem cells (PDMSCs) had been isolated by different protocols and applied in regenerative studies; however, the usage of PDMSCs in acute injury diseases were limited, which may due to therapeutic potential of those cells. Here, we demonstrated a novel serum-free selection culture condition to isolate MSCs from the chorionic membrane of the human term placenta, named pcMSC. In our preliminary studies, pcMSCs were used as therapeutic cells to treat paraquat induced acute lung injury in animal models. After the therapy, the results showed that at day-6, the survival rates of mice increased from 8% to 35% and decreased the infiltration of neutrophils into the lung tissues and alveolar spaces; however, other therapies, including treatment with dexamethasone and other cell types did not increase the survival rates. These results indicate that pcMSC cell therapy significantly restored lung function. Hence, we hypothesize that pcMSC can decrease neutrophils infiltration in the lung tissues and alveolar spaces. By flow cytometry analysis, the results showed that pcMSC was positive for MSC markers CD70, CD90, CD105, and CD29, CD44 but negative for hematopoietic lineage markers including CD14, CD34, and CD45. In addition to the above markers, pcMSC was positive for HLA-G. In further study, RT-PCR results indicate pcMSC express different isoforms of HLA-G, including membrane forms (HLA-G1, -G2, -G3, -G4) and soluble forms (HLA-G5, and –G6). Moreover, immunohistochemistry (IHC) and immunofluorescence (IFC) results of the human term placenta show that HLA-G was strongly expressed on the chorionic membrane and this protein was co-localized at the position where CD105 was expressed, which was on the chorionic membrane of the human term placenta. In order to reveal the roles of HLA-G in immunoregulation of neutrophils, a neutrophil-like HL60 cell was used to study the interaction between HLA-G and neutrophils. This interaction of HLA-G and differentiated HL60 cells were examined using a co-culture migration system. The results showed pcMSC down-regulated the migration of differentiated HL-60 cells. To verify HLA-G was the main molecule in this down-regulation effect, a neutralizing antibody was used to block HLA-G and the results showed that HLA-G is an important molecule that inhibits the migration of differentiated HL-60 cells. This result proposes that pcMSC may be used as a model to further understand the immunomodulatory effects between HLA-G and neutrophils. Furthermore, these results show that pcMSC may be a promising cell used in cell therapies for diseases with acute injury.
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