脐带间充质干细胞培养及体外重建角膜后板层的初步研究

发布时间：2018-09-09 20:25

【摘要】： 目的(1)应用改良及传统培养方法体外培养兔角膜基质细胞和人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs),观察其生物学特性,为下一步角膜后板层重建奠定基础。(2)探讨以板层猪角膜基质后弹力层面为载体培养人脐带间充质干细胞初步构建组织工程角膜后板层的可行性,为组织工程角膜后板层的构建提供新的种子细胞来源和适宜的载体材料。方法(1)采用新培养法—悬浮基质片法及传统方法对兔角膜基质细胞进行分离、培养及鉴定。(2)采用新培养法—脐带组织片悬浮法及传统方法对人脐带MSCs进行分离、纯化、扩增、传代,观察其体外生长特征。(3)采用流式细胞术对体外培养的人脐带MSCs进行免疫表型鉴定,并行成脂细胞和成骨细胞诱导分化,鉴定其干细胞特性。(4)将第五代脐带MSCs接种于去除内皮细胞的板层猪角膜基质后弹力层面上进行培养。(5)待细胞融合形成单层后,观察接种后细胞的生长特性,3周后行HE染色及扫描电镜、透射电镜等对细胞进行形态学观察。免疫组化法检测接种后干细胞角膜内皮细胞抗原标志物神经元特异性烯醇化酶(NSE)的表达。结果(1)新的培养方法—悬浮基质片法及脐带组织片悬浮法可成功培养出角膜基质细胞及脐带间充质干细胞,较传统方法便捷、可靠。体外培养的角膜基质细胞及脐带MSCs均呈成纤维细胞形态,增殖活力好。(2)流式细胞仪示：培养的人脐带MSCs表达间充质干细胞的特异标记包括CD13、CD29、CD44、CD105,但不表达造血和内皮细胞的标志CD31、CD34、CD45。经体外诱导证实,人脐带MSCs具有向脂肪细胞及成骨细胞方向分化的能力。(3)将传代后的人脐带MSCs接种于板层猪角膜基质后弹力层面上进行培养,细胞可在后弹力层面贴附,单层生长良好；电镜下见细胞表面微绒毛丰富,细胞之间紧密连接,富含细胞器。经培养3周后部分干细胞表达角膜内皮细胞抗原标志物神经元特异性烯醇化酶(NSE)。结论(1)本研究探讨了兔角膜基质细胞和人脐带MSCs体外分离、培养的改良方法,体外培养的角膜基质细胞和脐带MSCs均呈成纤维细胞形态,具有较强增殖能力。(2)体外培养的人脐带间充质干细胞,具有多向分化潜能。以板层猪角膜基质后弹力层面为载体可成功培养人脐带间充质干细胞,构建出与正常角膜后板层相似的组织,有望为组织工程角膜后板层重建提供一种新的种子细胞来源和适宜的载体材料,并为进一步深入进行功能性研究和移植提供了实验基础。
[Abstract]:Objective (1) to observe the biological characteristics of rabbit corneal stromal cells and human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUCMSCs),) cultured in vitro by modified and traditional methods. (2) to explore the feasibility of using the lamellar layer of porcine corneal stroma as the carrier to culture human umbilical cord mesenchymal stem cells to construct the tissue engineered posterior lamina of cornea. To provide a new seed cell source and suitable carrier material for the construction of the posterior lamina of tissue engineering cornea. Methods (1) the rabbit corneal stromal cells were isolated, cultured and identified by the new culture-suspension matrix method and the traditional method. (2) the human umbilical cord MSCs was isolated, purified and amplified by the new culture-umbilical cord tissue suspension method and the traditional method. (3) Immunophenotypes of human umbilical cord MSCs cultured in vitro were identified by flow cytometry and induced by adipoblasts and osteoblasts. (4) the fifth generation of umbilical cord MSCs was inoculated on the elastic layer of the lamellar porcine corneal stroma to remove endothelial cells. (5) when the cells were fused into monolayers, The growth characteristics of the cells were observed 3 weeks after inoculation. HE staining, scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the cells. The expression of neuron-specific enolase (NSE) was detected by immunohistochemistry. Results (1) the corneal stromal cells and umbilical cord mesenchymal stem cells could be successfully cultured by a new culture method-suspension matrix method and umbilical cord tissue suspension method, which was more convenient and reliable than the traditional method. Corneal stromal cells and umbilical cord MSCs cultured in vitro showed fibroblast morphology and good proliferative activity. (2) flow cytometry showed that the specific markers of cultured human umbilical cord MSCs expressing mesenchymal stem cells included CD13,CD29,CD44,CD105, but not hematopoietic and endothelial cell markers CD31,CD34,CD45. Human umbilical cord MSCs had the ability to differentiate into adipocytes and osteoblasts by induction in vitro. (3) the cultured human umbilical cord MSCs was cultured on the elastic layer of lamellar porcine corneal stroma, and the cells could be attached to the posterior elastic layer. The growth of monolayer was good. Under electron microscope, the microvilli on the surface of the cells were abundant, the cells were tightly connected and the organelles were rich. After cultured for 3 weeks, some stem cells expressed neuron-specific enolase (NSE). As a marker of corneal endothelial cell antigen. Conclusion (1) the improved method of isolation and culture of rabbit corneal stromal cells and human umbilical cord MSCs in vitro was studied. The corneal stromal cells and umbilical cord MSCs in vitro were fibroblast morphology. (2) cultured human umbilical cord mesenchymal stem cells have the potential of multiple differentiation. Human umbilical cord mesenchymal stem cells could be cultured successfully using the lamellar porcine corneal stromal layer as the carrier, and the tissue similar to the normal corneal posterior lamina could be constructed. It is expected to provide a new source of seed cells and a suitable carrier material for the reconstruction of the posterior lamina of tissue engineering cornea and provide experimental basis for further functional research and transplantation.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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