论文标题:脐带干细胞的分离、鉴定及分化为心肌和内皮细胞的实验研究
The Isolation, Characterization of Human Umbilical Cord Derived Stem Cells and Its Differentiation Potential to Myocardial and Endothelial Cells
论文作者
论文导师 刘迎龙,论文学位 博士,论文专业 外科学
论文单位 中国协和医科大学,点击次数 135,论文页数 129页File Size12776K
2007-05-01论文网 http://www.lw23.com/lunwen_242093897/
Umbilical cord derived stem cells;; isolation;; characterization;; differentiation;; cardiomyocytes;; endothelial cells
心血管疾病严重威胁人类的健康，细胞移植或体外构建组织工程移植物将是一个很有前景的治疗选择。但组织工程移植物面临再血管化或内皮化的问题，最近研究发现干细胞在体外可以分化为心肌和内皮细胞，对细胞移植和构建组织工程移植物有非常重要的意义。骨髓间充质干细胞是研究最多的干细胞，但是随着年龄的增加，骨髓间充质干细胞的数量和体外增殖分化能力都明显减弱，这需要我们去寻找更好的干细胞来源，脐带组织是非常有希望的干细胞替代来源。 本研究的第一部分采用胶原酶消化法从脐带组织中分离干细胞，新鲜分离的脐带细胞在培养72h后，开始贴壁，5天后贴壁细胞开始增多，形成细胞克隆，传代培养，细胞逐渐纯化，形态呈均一的细长梭形。流式细胞仪分析这些细胞表达CD13，CD29，CD44，CD90，CD166和MHC-Ⅰ，而不表达造血和内皮细胞的标志物，如CD31、CD34、CD38、CD45、CD106、CD117，CD144和MHC-Ⅱ，经多次传代后，表面分子表达无明显改变。在成骨细胞和成脂肪细胞诱导培养后，VonKossa染色和油红O染色阳性，说明其能够分化为骨细胞和脂肪细胞。这说明我们分离的脐带细胞与骨髓间充质干细胞具有相似的特性，证明了其干细胞特性，我们称之为UCDS细胞。 在第二部分中，我们研究了在5-氮胞苷的作用下，UCDS细胞能否分化为心肌样细胞。5-氮胞苷诱导后，细胞的形态不断发生变化，但是没有观察到明显的肌管形成，也没有观察到可以跳动的心肌细胞。RT-PCR检测显示分化后的UCDS细胞表达心肌特异性基因肌钙蛋白T，免疫组化检测诱导后的细胞表达心肌特异性α-肌动蛋白、肌球蛋白和肌钙蛋白T，表达率在50％以上。电镜下观察，诱导4周后，UCDS细胞有明显的肌丝样结构形成，说明在5-氮胞苷的作用下，UCDS细胞能够分化为心肌样细胞。 第三部分的目的是研究在内皮细胞生长因子和碱性成纤维细胞生长因子的作用下，UCDS细胞能否向内皮细胞方向分化。在体外经过诱导之后，细胞形态不断发生变化，形成网格样结构。免疫荧光显示，分化后的UCDS细胞CD31、CD34染色阳性，具有摄取DiI-Ac-LDL的功能。流式鉴定细胞的分化效率可以达到50％左右。下肢缺血动物模型也证明，UCDS细胞能够在体内缺血环境下分化为内皮细胞，并且参与血管新生，改善下肢缺血状况。我们的研究结果表明UCDS细胞是非常有应用前景的干细胞来源，为心肌细胞移植和体外构建组织工程移植物以及移植物再血管化研究提供了新的种子细胞选择。
Abstract: Cardiovascular diseases are a leading cause of morbidity and mortality. Currently, the valves, patches and conduits for replacement therapy or repair are imperfect and subject patients to one or more ongoing risks including thrombosis, limited durability, increased susceptibility to infection, and need for reoperations due to lack of growth in pediatric populations. A very promising approach to solve the above problems may be the use of tissue engineering with autologous cells. The successful use of tissue-engineered transplants is hampered by the need for vascularization. Recent advances have made possible the using of stem cells as cell sources for therapeutic angiogenesis, including the vascularization of engineered tissue grafts. Stem cells are undifferentiated cells capable of self-renewal and differentiation into multiple lineages of mature cells. Mesenchymal stem cells (MSCs) can be isolated from various adult tissues of human by their ability of adhering plastic culture plate wall. Despite the fact that bone marrow derived MSCs represent the main available source for cell therapies, the use of bone marrow derived MSCs is not always acceptable because of the significant decrease in cell number and proliferation/differentiation capacity with age. In addition, obtaining the therapeutic quantity of bone marrow requires general anesthesia and hospitalization. In this connection, most attention should be paid to tissues containing cells with higher proliferative potency, capability of differentiation, and low risk of contamination. Human umbilical cord is likely a feasible source of stem cells for its advantage over bone marrow such as vast abundance, lack of donor attrition, and low risk of viral transmission. In the first part of this paper, we described the isolation and characterization of stem cells from human umbilical cord tissue (UCDS cells). When initially plated, the UCDS cells appeared rounded in shape. After 72h of plating, the cells were adherent, elongated, and spindle-shaped. Flow cytometry results showed that UCDS cells were positive for CD13, CD29, CD44, CD90, CD166, and MHC-I, in addition, no expression of CD31, CD34, CD38, CD45, CD106, CD117 and CD144 or MHC-Ⅱwas observed. Most importantly, the cells were positive for Kossa staining and have the ability to accumulate different amounts of lipid vacuoles after cultivation in osteogenic and adipogenic medium. This results demonstrated that UCDS cells were a crowd of undifferentiated stem cells that were similar to bone marrow derived MSCs, have similar phenotype and differentiation ability. In the second part, we investigated the myocardial differentiation ability of UCDS cells. Two weeks after treatment. Some cells gradually increased in size and formed a ball-like or stick-like appearance, but we have not observed beating cells during the in vitro differentiation. RT-PCR results showed that differentiated cells express cardiomyocyte Specific cardiac troponin T gene. Immunocytochemistry showed that differentiated cells were strongly stained with cardiac sarcomericα-actin myosin and Troponin T at 1 month after 5-azacytidine treatment and more than 50% UCDS cells were positively stained. Longitudinal sections of UCDS cells which were directed toward cardiomyocytes were analyzed by transmission electron microscopy. After 4 weeks of induction with 5-azacytidine, some cells showed myofilaments, but their alignment are intricate. The goal of the third part of our study was to examine the endothelial potential of UCDS cells. UCDS cells were differentiated in an endothelial differentiation medium containing VEGF and bFGF. Differentiation into endothelial cells was determined by acetylated low-density lipoprotein (ac-LDL) incorporation and expression of endothelial-specific proteins. The uptake of DiI-labeled ac-LDL is a specific marker for endothelial cells in vitro. Immunofluorescence revealed that the induced cells were positive for ac-LDL uptake. Immunofluorescence studies confirmed their endothelial phenotype with expression of known endothelial cell markers including CD31, CD34 and 30-50% UCDS cells were positively stained. In addition, different staining from cells in the same area of culture showed the induced cells which were positive for Ac-LDL uptake were also positively stained with CD31 and CD34. In vivo, the transplanted UCDS cells were sprouting from local injection and differentiated into endothelial cells in a hindlimb ischemia mouse model. These findings indicate the presence of a cell population within the human umbilical cord that exhibits characteristics of endothelial progenitor cells. Therefore, human umbilical cord might represent a source of stem Cells useful for therapeutic angiogenesis and re-endothelialization of engineered tissue grafts. Umbilical cord derived stem cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous Use even if there was no immediate indication that stem cell therapy would be required. In the near future, cryopreserved autologous UCDS cells for therapeutic medicine may become available.
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