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標題: | 絨毛膜間質幹細胞中HLA-G蛋白與類嗜中性白血球HL60作用之探討 Regulation of HL60 Cells by HLA-G Molecules on Placenta Chorion Mesenchymal Stem Cells (pcMSCs) |
作者: | Yi-Ting Hsieh 謝伊婷 |
指導教授: | 林泰元 |
關鍵字: | 絨毛膜間質幹細胞,類嗜中性白血球,HL60, Placenta Chorion Mesenchyma Stem Cells,Human Leukocyte Antigen-G,HL60, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 現今,中胚層幹細胞 (MSCs) 已廣泛應用於不同疾病之臨床試驗,相較於其傳統之來源:骨髓幹細胞,人類胎盤幹細胞為易於取得之替代來源。早先,依不同程序分離出來之胎盤衍生中胚層幹細胞 (PDMSCs) 已應用於再生醫學之研究。然而,PDMSCs在急性傷害之應用仍十分有限,可能受限於PDMSCs本身之治療潛力。本論文提出一套新穎的無血清培養條件、適用於人類胎盤絨毛膜中,中胚層幹細胞 (稱為pcMSCs) 之篩選和分離。首先,我們初步觀察到pcMSCs適用於動物模式之百草枯(paraquat)所引發之急性肺損傷,在pcMSCs治療六天後,小鼠存活率從8%提升至35%,嗜中性球在肺組織以及肺泡腔之浸潤亦有降低;反觀其他治療,例如固醇類藥物dexamethasone,或使用其他中胚層幹細胞,則無法提升存活率,說明pcMSCs可顯著恢復肺組織之生理功能。因此,我們提出了psMSCs可以減輕肺組織和肺泡腔中嗜中性球浸潤之假設。再者,流式細胞儀結果顯示,pcMSCs會表現MSCs markers例如CD70、CD90、CD105、CD29、CD44,以及HLA-G,但不表現造血細胞marker例如CD14、CD34、以及CD45。而進一步的研究指出,pcMSCs表現不同亞型之HLA-G,包括membrane form 之HLA-G1、G2、G3、G4,和soluble form之HLA-G5、G6、G7。此外,人類胎盤之免疫組織染色和免疫瑩光染色結果顯示,HLA-G於人類胚胎絨毛膜中有大量表現,並且同時存在於CD105所表現之位置。為了瞭解HLA-G在嗜中性球之調控所扮演的角色以及兩者的關係,我們使用和嗜中性球相似之HL-60 cell,並且用co-culture migration system來進行研究,結果顯示pcMSCs抑制HL-60 differentiated cell的移動,而HLA-G在此抑制作用扮演重要角色之結果,在使用neutralizing antibody阻斷HLA-G後得到進一步證實。綜合上述之結果,pcMSCs適用於急性損傷之治療,並且可做為研究HLA-G和嗜中性球兩者間免疫調控之細胞模式。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23453 |
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