两种羊膜细胞的免疫调节功能研究
发布时间：2018-12-31 14:38
【摘要】：目的： 成体干细胞是一种具有自我更新和多向分化能力的细胞，在组织工程、基因治疗和细胞移植领域具有较好的应用前景。近年研究显示，人羊膜中也存在两种干细胞：羊膜上皮细胞（human Amniotic epithelial cells， HAEC）和羊膜间充质细胞（human Amniotic mesenchymal cells，HAMC），由于其具有来源广泛、取材方便、多向分化潜能以及免疫原性低等优点，正逐渐成为干细胞研究领域的热点之一。本课题旨在通过研究HAEC及HAMC的免疫调节功能，从免疫学角度探讨其同种异体移植的可行性，探索两种羊膜细胞的免疫调节作用在预防及治疗移植物抗宿主病（graft versus-host disease，GVHD）等免疫性疾病的潜在价值。 方法： 1.体外分离培养两种人羊膜细胞，通过流式细胞术和免疫荧光法鉴定其表型差异。 2.通过对两种羊膜细胞条件诱导分化，鉴定其多项分化潜能。 3.将两种羊膜细胞及其上清液分别与淋巴细胞混合培养，用MTT检测其对淋巴细胞增殖能力的影响。 4.用流式细胞术检测羊膜细胞与淋巴细胞混合培养后T淋巴细胞亚群和细胞内因子的变化情况。 5.通过Transwell系统建立非接触共培养体系，探索免疫抑制功能的机制。 结果： 1.成功建立两种人羊膜细胞的体外分离培养和扩增方法。 2.两种羊膜细胞阳性表达HLA-A, B, C和间充质干细胞的标志（CD29, CD73,CD44, CD59, CD90, CD105, CH166），不表达造血干细胞的标志(CD31, CD34,CD45, HLA-DR)，弱表达协同刺激分子(CD40, CD40L, CD80,CD86)，，且这些表型的表达量在P3-P7代都维持稳定。 3.免疫荧光法显示HAEC表达角蛋白19，不表达波形蛋白，而HAMC正好相反。 4. HAMC更好地向心肌细胞分化，而HAEC更好地向神经细胞分化。 5.两种羊膜细胞及其上清液均能抑制淋巴细胞的增殖。 6.淋巴细胞与HAMC混合培养后，CD4+T细胞亚群增加，CD8+T细胞亚群显著减少；Th1和Tc1比率较对照组有显著降低，而Th2和Tc2的比率却有轻度上升，HAEC的作用无显著性差异。 7.通过Transwell系统发现两种羊膜细胞均可以分泌可溶性细胞因子，发挥免疫抑制功能。 结论： 从羊膜中可分离出HAEC和HAMC，且两种细胞的表型相似，多向分化潜能却不同。HAEC具有更好地外胚层分化潜能，而HAMC能更好地向中胚层分化。两种羊膜细胞均可分泌可溶性细胞因子，发挥免疫抑制功能。HAMC在体外明显抑制T淋巴细胞的增殖，尤其是CD8+T细胞。它还能降低MLC体系中的Th1和Tc1,升高Th2和Tc2，所以在临床上可以减轻抑制后急性移植物抗宿主病（aGVHD）的发生，而保留移植物抗白血病（GVL）的潜力。HAEC同样具有免疫抑制作用，由于其作用机制的不同，更适合作为组织修复工程的种子细胞。
[Abstract]:Objective: adult stem cells are self-renewing and multi-differentiated cells, which have a good prospect in tissue engineering, gene therapy and cell transplantation. Recent studies have shown that there are two kinds of stem cells in human amniotic membrane: amniotic epithelial cells (human Amniotic epithelial cells, HAEC) and amniotic mesenchymal cells (human Amniotic mesenchymal cells,HAMC). The advantages of multiple differentiation potential and low immunogenicity are becoming one of the hotspots in stem cell research. The purpose of this study is to study the immunomodulatory function of HAEC and HAMC, to explore the feasibility of allograft transplantation from the immunological point of view, and to explore the role of the two kinds of amniotic cells in the prevention and treatment of graft-versus-host disease (graft versus-host disease,). The potential value of immune diseases such as GVHD. Methods: 1. Two kinds of human amniotic membrane cells were isolated and cultured in vitro, and their phenotypic differences were identified by flow cytometry and immunofluorescence. 2. The differentiation potential of two kinds of amniotic cells was identified by inducing the differentiation of two kinds of amniotic cells. 3. The two kinds of amniotic cells and their supernatants were mixed with lymphocytes respectively and the effects of the two kinds of amniotic cells and their supernatants on lymphocyte proliferation were detected by MTT. 4. The changes of T lymphocyte subsets and intracellular factors in amniotic membrane cells and lymphocytes were detected by flow cytometry. 5. A non-contact co-culture system was established by Transwell system to explore the mechanism of immunosuppressive function. Results: 1. Two methods of isolation, culture and amplification of human amniotic membrane cells in vitro were successfully established. 2. Two kinds of amniotic cells expressed HLA-A, B, C and CD29, CD73,CD44, CD59, CD90, CD105, CH166, but not hematopoietic stem cells (CD31, CD34,CD45, HLA-DR). The expression of co-stimulatory molecules (CD40, CD40L, CD80,CD86) was weakly expressed, and the expression of these phenotypes remained stable in P3-P7 generation. 3. Immunofluorescence assay showed that HAEC expressed keratin 19 and did not express vimentin, whereas HAMC expressed the opposite. 4. HAMC differentiates better into cardiomyocytes, while HAEC differentiates better into neural cells. 5. Both amniotic cells and their supernatants could inhibit the proliferation of lymphocytes. 6. After mixed culture of lymphocytes and HAMC, CD4 T cell subsets increased, CD8 T cell subsets decreased significantly, Th1 and Tc1 ratios decreased significantly, but Th2 and Tc2 ratios increased slightly. HAEC had no significant difference. 7. It was found by Transwell system that both kinds of amniotic cells could secrete soluble cytokines and exert immunosuppressive function. Conclusion: HAEC and HAMC, can be isolated from amniotic membrane. The phenotypes of the two cells are similar, but the multidirectional differentiation potential is different. HAEC has better ectodermal differentiation potential, while HAMC can better differentiate into mesoderm. Both kinds of amniotic cells secreted soluble cytokines and played an immunosuppressive role. HAMC significantly inhibited the proliferation of T lymphocytes, especially CD8 T cells in vitro. It can also reduce the increase of Th1 and Tc1, in MLC system and increase Th2 and Tc2, so it can attenuate the occurrence of (aGVHD) in acute graft-versus-host disease after inhibition. HAEC also has immunosuppressive effect, because of its different mechanism, HAEC is more suitable as seed cells for tissue repair engineering.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.4
[Abstract]:Objective: adult stem cells are self-renewing and multi-differentiated cells, which have a good prospect in tissue engineering, gene therapy and cell transplantation. Recent studies have shown that there are two kinds of stem cells in human amniotic membrane: amniotic epithelial cells (human Amniotic epithelial cells, HAEC) and amniotic mesenchymal cells (human Amniotic mesenchymal cells,HAMC). The advantages of multiple differentiation potential and low immunogenicity are becoming one of the hotspots in stem cell research. The purpose of this study is to study the immunomodulatory function of HAEC and HAMC, to explore the feasibility of allograft transplantation from the immunological point of view, and to explore the role of the two kinds of amniotic cells in the prevention and treatment of graft-versus-host disease (graft versus-host disease,). The potential value of immune diseases such as GVHD. Methods: 1. Two kinds of human amniotic membrane cells were isolated and cultured in vitro, and their phenotypic differences were identified by flow cytometry and immunofluorescence. 2. The differentiation potential of two kinds of amniotic cells was identified by inducing the differentiation of two kinds of amniotic cells. 3. The two kinds of amniotic cells and their supernatants were mixed with lymphocytes respectively and the effects of the two kinds of amniotic cells and their supernatants on lymphocyte proliferation were detected by MTT. 4. The changes of T lymphocyte subsets and intracellular factors in amniotic membrane cells and lymphocytes were detected by flow cytometry. 5. A non-contact co-culture system was established by Transwell system to explore the mechanism of immunosuppressive function. Results: 1. Two methods of isolation, culture and amplification of human amniotic membrane cells in vitro were successfully established. 2. Two kinds of amniotic cells expressed HLA-A, B, C and CD29, CD73,CD44, CD59, CD90, CD105, CH166, but not hematopoietic stem cells (CD31, CD34,CD45, HLA-DR). The expression of co-stimulatory molecules (CD40, CD40L, CD80,CD86) was weakly expressed, and the expression of these phenotypes remained stable in P3-P7 generation. 3. Immunofluorescence assay showed that HAEC expressed keratin 19 and did not express vimentin, whereas HAMC expressed the opposite. 4. HAMC differentiates better into cardiomyocytes, while HAEC differentiates better into neural cells. 5. Both amniotic cells and their supernatants could inhibit the proliferation of lymphocytes. 6. After mixed culture of lymphocytes and HAMC, CD4 T cell subsets increased, CD8 T cell subsets decreased significantly, Th1 and Tc1 ratios decreased significantly, but Th2 and Tc2 ratios increased slightly. HAEC had no significant difference. 7. It was found by Transwell system that both kinds of amniotic cells could secrete soluble cytokines and exert immunosuppressive function. Conclusion: HAEC and HAMC, can be isolated from amniotic membrane. The phenotypes of the two cells are similar, but the multidirectional differentiation potential is different. HAEC has better ectodermal differentiation potential, while HAMC can better differentiate into mesoderm. Both kinds of amniotic cells secreted soluble cytokines and played an immunosuppressive role. HAMC significantly inhibited the proliferation of T lymphocytes, especially CD8 T cells in vitro. It can also reduce the increase of Th1 and Tc1, in MLC system and increase Th2 and Tc2, so it can attenuate the occurrence of (aGVHD) in acute graft-versus-host disease after inhibition. HAEC also has immunosuppressive effect, because of its different mechanism, HAEC is more suitable as seed cells for tissue repair engineering.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.4
【参考文献】
相关期刊论文 前2条
1 刘庆国,姜尔烈,黄勇,周世勇,王荷花,何垎,王志东,王玫,周征,翟文静,冯四洲,韩明哲;T细胞亚群Ⅰ和Ⅱ型细胞比例与急性移植物抗宿主病相关性研究[J];临床血液学杂志;2005年05期
2 宣e
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