胎盘来源多能细胞的体外分离培养与生物学特性研究

发布时间：2018-08-29 09:23

【摘要】： 目的间充质干细胞(mesenchymal stem cells，MSCs)具有高度增殖、自我更新的能力，广泛分布于多种人体组织中，参与机体组织器官的正常功能活动和损伤修复，是再生医学领域研究的热点。目前已成功地从人骨髓、外周血、肌肉、脂肪、脐血、羊水及胎儿组织中分离并鉴定出MSCs。其中骨髓来源间充质干细胞(bone marrow mesenchymal stemcells，BMSCs)研究最多，也取得了一些进展。但也发现临床采集骨髓有一定困难，可能感染并引起并发症，而且随着供者年龄增加其数量、增殖和分化能力均显著下降。近几年来有学者发现胎盘组织中存在MSCs。与骨髓取材相比胎盘在胎儿娩出后即完成使命，成为“废弃”物，其取材方便，易于分离，对其研究不会涉及伦理道德问题，因而可能成为组织修复和基因治疗的新细胞来源。本课题旨在探索体外分离培养胎盘来源多能细胞(placenta-derived mutipotent cells,PDMCs)的方法和条件，以及对其生物学和功能上的特性进行初步研究。对象和方法 (1)胎盘组织30份：在无菌条件下，采集足月健康顺产分娩孕妇的胎盘。 (2)PDMCs的制备：剪切胎盘蜕膜组织1~2mm~3大小的组织块，用酶消化法从中分离得到细胞悬液，通过人淋巴分离液密度梯度离心得到单核细胞，接种于培养瓶中。 (3)取原代到15代细胞分别利用MTT染色技术，免疫组织化学染色，HE染色，透射电镜，以及流式细胞检测仪，从不同层面和角度鉴定胎盘来源多能细胞的生物学特征，并与BMSCs进行对比研究，以观察两者之间在生物学特性方面的异同。结果 (1)细胞培养：通过混合酶消化法可以得到PDMCs，细胞形态良好，传至15代没有形态改变。 (2)细胞形态：通过倒置显微镜和HE染色观察可见细胞呈长梭形，漩涡状生长，细胞体积较大。 (3)细胞生长规律：原代细胞接种后的9-14天出现细胞克隆。传代之后的第4天为对数生长期，此后到第7天为平稳期。14天左右为衰退期。与文献报道的来自于骨髓的间充质干细胞的生长规律类似。 (4)细胞超微结构：通过透射电镜观察PDMCs和BMSCs，发现两者有共同的特点，即核膜不规则，核仁明显，核内以常染色质为主，且分布均匀；胞浆内，可见丰富的粗面内质网，线粒体，，核糖体等细胞器，并且胞膜完整，表面有大量微绒毛。 (5)表面标志：通过免疫组织化学染色和流式细胞检测，发现PDMCs表达CD105，CD166，CD44，CD29，CD9，HLA-ABC，不表达CD34，CD40L，和HLA-DR。这与BMSCs表面抗原标志表达相类似。PDMCs表达胚胎干细胞表面抗原标志SSEA-3，SSEA-4，TRA-1-60，TRA-1-81，Oct-4的量高于BMSCs。结论(1)用酶消化法可以从足月分娩的胎盘蜕膜组织中分离获得长梭形贴壁生长的细胞(PDMCs)，这类细胞具有与BMSCs相似的生物学特性。 (2)PDMCs较BMSCs更为原始
[Abstract]:Objective Mesenchymal stem cells (mesenchymal stem cells,MSCs) have the ability of high proliferation and self-renewal. They are widely distributed in a variety of human tissues and participate in the normal function and repair of tissues and organs, which is a hot research topic in the field of regenerative medicine. MSCs. has been successfully isolated and identified from human bone marrow, peripheral blood, muscle, fat, umbilical cord blood, amniotic fluid and fetal tissues. Bone marrow derived mesenchymal stem cells (bone marrow mesenchymal stemcells,BMSCs) have been studied most and some progress has been made. But it is also found that it is difficult to collect bone marrow in clinic, which may cause infection and complications, and with the increase of donor age, the ability of proliferation and differentiation are significantly decreased. In recent years, some scholars have found that MSCs. exists in placenta tissue. Compared with bone marrow, placenta completes its mission and becomes an "abandoned" object. It is convenient and easy to separate. The study of placenta is not related to ethical and moral issues, so it may become a new cell source for tissue repair and gene therapy. The aim of this study was to explore the methods and conditions of isolation and culture of placental pluripotent cells (placenta-derived mutipotent cells,PDMCs) in vitro and to study their biological and functional characteristics. Objects and methods (1) 30 samples of placental tissue: under aseptic conditions, The placenta of pregnant women with full term healthy delivery was collected. (2) the preparation of PDMCs: the tissue blocks of 1~2mm~3 in decidual tissue of placenta were cut off, and the cell suspension was isolated by enzyme digestion. Mononuclear cells were obtained by density gradient centrifugation of human lymphoid isolate and inoculated in culture flask. (3) Primary to 15th passage cells were stained by MTT staining, immunohistochemical staining with HE staining, transmission electron microscopy (TEM). And flow cytometry was used to identify the biological characteristics of placental pluripotent cells from different levels and angles, and to compare with BMSCs to observe the similarities and differences of biological characteristics between them. Results (1) Cell culture: PDMCs, cells could be obtained by mixed enzyme digestion. No morphological changes were observed at the 15th generation. (2) the morphology of cells was observed by inverted microscope and HE staining. (3) Law of cell growth: cell clone appeared 9-14 days after primary cell inoculation. The logarithmic growth period was on the 4th day after passage and the decline period was about 14 days from the 7th day. The growth pattern of mesenchymal stem cells from bone marrow was similar to that reported in the literature. (4) Ultrastructure of cells: PDMCs and BMSCs, showed the same characteristics by transmission electron microscope, that is, irregular nuclear membrane. In the cytoplasm, there are abundant organelles such as rough endoplasmic reticulum, mitochondria, ribosome and so on, and the cell membrane is intact. A large number of microvilli were found on the surface. (5) Surface markers: by immunohistochemical staining and flow cytometry, it was found that PDMCs expressed CD105,CD166,CD44,CD29,CD9,HLA-ABC, and did not express CD34,CD40L, and HLA-DR.. This is similar to the expression of BMSCs surface antigen marker. PDMCs express more embryonic stem cell surface antigen marker SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,Oct-4 than BMSCs.. Conclusion (1) long fusiform adherent cell (PDMCs), can be obtained from placental decidua tissue of term delivery by enzyme digestion. (2) PDMCs is more primitive than BMSCs.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

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