以羊膜为载体兔角膜缘上皮细胞的体外培养实验研究

发布时间：2018-01-19 02:40

本文关键词： 羊膜角膜缘上皮细胞培养　出处：《大连医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 研究背景:眼表疾病是眼科常见病,其中许多疾病都存在角膜缘干细胞缺陷或功能障碍,治疗也相当棘手。基于角膜缘干细胞的新概念,近10年来眼表面重建研究集中于角膜缘部上皮细胞,成为当今国际眼科研究热点之一。自体角膜缘上皮移植已在临床上广泛开展并取得良好的疗效。然而,由于自体取材在很多情况下受限,同种异体角膜缘上皮移植和以体外培养的角膜缘干细胞移植是最新的研究目标。目的:建立以羊膜为载体兔角膜缘上皮细胞的体外培养方法,了解其生物学特性,为最终实现以体外培养的兔角膜缘干细胞移植提供实验依据。方法:选择新鲜的兔眼球,在净化台内,用含有“双抗”的生理盐水进行冲洗后,制备兔角膜缘组织,将其剪切成约1mm×1mm大小的组织块。以去除上皮细胞的羊膜为载体,采用三种方法对兔角膜缘上皮细胞(limbal epithelial cell ,LEC)进行接种培养,分别为:组织块直接接种培养法、组织块制备单细胞培养法、组织块经酶消化处理后培养法。培养基分别采用了RPMI-1640和DMEM+Ham-F12,并添加了相同的营养因子。倒置显微镜下观察培养细胞体外生长的形态。结果:1、组织块直接接种培养法:接种培养第3天,上皮细胞开始从角膜缘组织块周围移行到羊膜上,形成典型的“沙滩样”移行带。第5～7天,大部分组织块上的细胞从移行带向外生长形成生长晕,相互连接成膜状。第13～15天,细胞汇合在一起,排列紧密,铺满整个羊膜。2、组织块制备单细胞培养法:培养接种5天后上皮细胞开始在羊膜上贴壁,细胞呈圆形,大小一致。第8天左右细胞开始生长,2周时细胞汇合在一起,形成网状结构。同时,生长的上皮细胞中有成纤维样细胞的混杂生长,但各自保持其良好的细胞形态。3周左右形成良好的单层,细胞生长呈膜片状扩展,整个羊膜之上铺满细胞,有明显的扩展界限。但羊膜之上有两种细胞混合生长,成纤维细胞的生长明显强于上皮细胞。3、组织块经酶消化处理后培养法:培养第7天,部分上皮细胞开始在羊膜上贴壁,但大多以散在形式生长。在部分细胞贴壁的同时,培养液中有悬浮生长的细胞,换液后仍然存在。第15天,羊膜上贴壁的细胞逐渐增多,但大多数以单细胞生长,也可见少量的细胞团。第20～25天左右,培养的细胞汇合在一起,形成团状结构,逐渐成网状后连接成片,但不能形成良好的单层。结论:1、羊膜是兔LEC体外培养的良好载体。2、在以羊膜为载体的三种接种培养方法中(①组织块直接接种培养法;②组织块制备单细胞培养法;③组织块经酶消化处理后培养法),组织块直接接种培养法效果最佳。3、以RPMI-1640和DMEM+ Ham-F12(体积比3:1)为培养基培养兔LEC,在培养基中添加相同的营养成份,培养结果无明显差异。
[Abstract]:Background: ocular surface disease is a common ophthalmic disease, many of which have corneal limbal stem cell defects or dysfunction, treatment is very difficult. Based on the new concept of limbal stem cells. In the past 10 years, the study of ocular surface reconstruction has focused on limbal epithelial cells, which has become one of the international ophthalmic research hotspots. Limbal autograft transplantation has been widely used in clinic and has achieved good results. Autogenous limbal epithelium transplantation and limbal stem cell transplantation in vitro are the latest research objectives. Aim: to establish an in vitro culture method of rabbit corneal limbal epithelial cells with amniotic membrane as carrier, to understand its biological characteristics, and to provide experimental evidence for the ultimate transplantation of rabbit limbal stem cells in vitro. Methods: the rabbit limbal tissue was prepared by washing the fresh rabbit eyeball with saline containing "double antibodies" in the purifying table. It was cut into a tissue about 1 mm 脳 1 mm in size. The amniotic membrane was used as the carrier to remove the epithelial cells. Rabbit limbal epithelial cell were cultured by three methods: direct inoculation of tissue mass. Single cell culture method was used to prepare tissue block, and enzyme digestion was used to culture tissue block. RPMI-1640 and DMEM Ham-F12 were used in culture medium. The growth of cultured cells in vitro was observed under inverted microscope. Results 1. Direct inoculation of tissue mass: on the third day of inoculation, the epithelial cells began to migrate from the limbal tissue to the amniotic membrane, forming a typical "beach like" transition zone on the 5th day. Most of the cells on the tissue mass grew outwards from the transitional zone to form a growth halo, linked to each other to form a membrane. On the 13th 15th day, the cells converged and arranged tightly, covering the whole amniotic membrane .2. Tissue block preparation single cell culture method: after 5 days of culture, epithelial cells began to adhere to the amniotic membrane, the cells were round and the size was the same. On the 8th day, the cells began to grow and converge together for 2 weeks. At the same time, fibroblast-like cells in the growth of epithelial cells mixed growth, but each kept its good cell morphology about 3 weeks to form a good monolayer, cell growth as a lamellar expansion. The whole amniotic membrane was covered with cells with obvious expansion boundary. However, there were two kinds of cells on the amniotic membrane which were mixed and the growth of fibroblasts was stronger than that of epithelial cells. 3. The method of tissue mass culture by enzyme digestion: on the 7th day of culture, some epithelial cells began to adhere to the amniotic membrane, but most of them grew in the form of scattered. While some cells adhered to the wall, there were suspended growth cells in the culture medium. On the 15th day, the cells attached to the amniotic membrane gradually increased, but most of them grew as single cells, and a small number of cell clusters could also be seen. On the 20th and 25th day, the cultured cells converged together. Forming a mass structure, gradually forming a network after joining into a piece, but can not form a good monolayer. Conclusion amniotic membrane is a good carrier for rabbit LEC culture in vitro. (2) preparation of tissue blocks by single cell culture; 3The culture method of tissue block was digested by enzyme, and the method of direct inoculation of tissue mass was the best. 3. RPMI-1640 and DMEM Ham-F12 (volume ratio 3: 1) were used to culture rabbit RPMI-1640.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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