【摘要】：目的： 成體干細(xì)胞是一種具有自我更新和多向分化能力的細(xì)胞，在組織工程、基因治療和細(xì)胞移植領(lǐng)域具有較好的應(yīng)用前景。近年研究顯示，人羊膜中也存在兩種干細(xì)胞：羊膜上皮細(xì)胞（human Amniotic epithelial cells， HAEC）和羊膜間充質(zhì)細(xì)胞（human Amniotic mesenchymal cells，HAMC），由于其具有來源廣泛、取材方便、多向分化潛能以及免疫原性低等優(yōu)點，正逐漸成為干細(xì)胞研究領(lǐng)域的熱點之一。本課題旨在通過研究HAEC及HAMC的免疫調(diào)節(jié)功能，從免疫學(xué)角度探討其同種異體移植的可行性，探索兩種羊膜細(xì)胞的免疫調(diào)節(jié)作用在預(yù)防及治療移植物抗宿主�。╣raft versus-host disease，GVHD）等免疫性疾病的潛在價值。 方法： 1.體外分離培養(yǎng)兩種人羊膜細(xì)胞，通過流式細(xì)胞術(shù)和免疫熒光法鑒定其表型差異。 2.通過對兩種羊膜細(xì)胞條件誘導(dǎo)分化，鑒定其多項分化潛能。 3.將兩種羊膜細(xì)胞及其上清液分別與淋巴細(xì)胞混合培養(yǎng)，用MTT檢測其對淋巴細(xì)胞增殖能力的影響。 4.用流式細(xì)胞術(shù)檢測羊膜細(xì)胞與淋巴細(xì)胞混合培養(yǎng)后T淋巴細(xì)胞亞群和細(xì)胞內(nèi)因子的變化情況。 5.通過Transwell系統(tǒng)建立非接觸共培養(yǎng)體系，探索免疫抑制功能的機制。 結(jié)果： 1.成功建立兩種人羊膜細(xì)胞的體外分離培養(yǎng)和擴增方法。 2.兩種羊膜細(xì)胞陽性表達HLA-A, B, C和間充質(zhì)干細(xì)胞的標(biāo)志（CD29, CD73,CD44, CD59, CD90, CD105, CH166），不表達造血干細(xì)胞的標(biāo)志(CD31, CD34,CD45, HLA-DR)，弱表達協(xié)同刺激分子(CD40, CD40L, CD80,CD86)，，且這些表型的表達量在P3-P7代都維持穩(wěn)定。 3.免疫熒光法顯示HAEC表達角蛋白19，不表達波形蛋白，而HAMC正好相反。 4. HAMC更好地向心肌細(xì)胞分化，而HAEC更好地向神經(jīng)細(xì)胞分化。 5.兩種羊膜細(xì)胞及其上清液均能抑制淋巴細(xì)胞的增殖。 6.淋巴細(xì)胞與HAMC混合培養(yǎng)后，CD4+T細(xì)胞亞群增加，CD8+T細(xì)胞亞群顯著減少；Th1和Tc1比率較對照組有顯著降低，而Th2和Tc2的比率卻有輕度上升，HAEC的作用無顯著性差異。 7.通過Transwell系統(tǒng)發(fā)現(xiàn)兩種羊膜細(xì)胞均可以分泌可溶性細(xì)胞因子，發(fā)揮免疫抑制功能。 結(jié)論： 從羊膜中可分離出HAEC和HAMC，且兩種細(xì)胞的表型相似，多向分化潛能卻不同。HAEC具有更好地外胚層分化潛能，而HAMC能更好地向中胚層分化。兩種羊膜細(xì)胞均可分泌可溶性細(xì)胞因子，發(fā)揮免疫抑制功能。HAMC在體外明顯抑制T淋巴細(xì)胞的增殖，尤其是CD8+T細(xì)胞。它還能降低MLC體系中的Th1和Tc1,升高Th2和Tc2，所以在臨床上可以減輕抑制后急性移植物抗宿主�。╝GVHD）的發(fā)生，而保留移植物抗白血病（GVL）的潛力。HAEC同樣具有免疫抑制作用，由于其作用機制的不同，更適合作為組織修復(fù)工程的種子細(xì)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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.4

【參考文獻】

相關(guān)期刊論文 前2條

1 劉慶國,姜爾烈,黃勇,周世勇,王荷花,何垎,王志東,王玫,周征,翟文靜,馮四洲,韓明哲;T細(xì)胞亞群Ⅰ和Ⅱ型細(xì)胞比例與急性移植物抗宿主病相關(guān)性研究[J];臨床血液學(xué)雜志;2005年05期

2 宣e

本文編號：2396721