大鼠表皮干细胞的分离培养及鉴定

发布时间：2018-03-07 04:17

本文选题：表皮干细胞　切入点：分离培养　出处：《西北农林科技大学》2009年硕士论文　论文类型：学位论文

【摘要】： 表皮干细胞(Epidermal Stem Cells,ESCs)是一类存在于皮肤组织中的多能性细胞,能够自我更新且具有多相分化潜能。本研究分别取成年SD大鼠和新生大鼠的背部皮肤,分离培养ESCs,并用IV型胶原进行纯化,最终获得ESCs,并对其生物学特性进行了研究。通过本研究,优化了大鼠表皮干细胞分离培养体系和鉴定方法,对建立大鼠表皮干细胞系有十分重要的现实意义,为下一步研究大鼠表皮干细胞转基因及分化奠定基础。本试验的具体内容为: 1.大鼠表皮干细胞的分离培养及纯化取大鼠背部皮肤,分别采取组织块法和酶消化法分离培养大鼠ESCs。组织块法制备细胞时,将皮肤剪成0.1cm×0.1cm的小块,基底面向下贴于培养皿中,添加培养液并隔天换液。组织块法制备表皮干细胞时,3~4d后从组织块边缘长出上皮样细胞,7~12d后细胞呈铺路石样生长,在上皮样细胞层上有小圆形细胞聚集呈集落样生长。培养至20 d,形成许多100~200个细胞组成的大克隆。酶消化法制备细胞时分别用两种酶消化组织块:2.0U/ml的中性蛋白酶消化过夜;0.25%胰酶+0.02%EDTA消化10min。酶消化法制备表皮干细胞时,4~5d后细胞呈铺路石样生长,培养至12~15d,形成克隆,但是死细胞较多。利用表皮干细胞对IV型胶原的快速黏附特性纯化表皮干细胞。传代培养时,采用室温消化法和冷消化法消化传代,结果证明冷消化5～6h为最佳消化时间而室温消化最佳时间为5min。组织块法和酶消化法均能得到表皮干细胞阳性集落且分别传至第8代和第6代。 2.大鼠表皮干细胞的鉴定从形态上观察,表皮干细胞体积较小、圆形,核质比大,能够形成克隆。免疫组化染色结果显示,ESCs表达p63、K15、β1-integrin、α6-integrin。
[Abstract]:Epidermal Stem cells (Epidermal Stem cells) are a kind of pluripotent cells in skin tissue, which can renew themselves and have the potential of multiphase differentiation. In this study, the dorsal skin of adult SD rats and newborn rats were obtained, respectively. ESCs were isolated and cultured and purified with type IV collagen. The biological characteristics of ESCs were studied. The isolation culture system and identification method of rat epidermal stem cells were optimized. It is of great practical significance to establish rat epidermal stem cell line and lay a foundation for further research on the transgenic and differentiation of rat epidermal stem cells. The specific contents of this experiment are as follows:. 1. Isolation, culture and purification of rat epidermal stem cells. The dorsal skin of rats was isolated and cultured by tissue block method and enzyme digestion method. When the cells were prepared by tissue block method, the skin was cut into pieces of 0.1 cm 脳 0.1 cm, and the substrate was attached to the petri dish facing down. When the epidermal stem cells were prepared by tissue mass method, the epithelioid cells grew from the edge of the tissue mass for 712 days, and the cells grew like paving stone after 4 days. In the epithelioid cell layer, small round cells gathered and grew like colony. After 20 days of culture, a large number of 100 ~ 200 cell clones were formed. When the cells were prepared by enzyme digestion, they were digested with two kinds of enzyme to digest the neutral protease of 1: 2.0Uml. After digesting 0.25% trypsin 0.02TA for 10 mins, the epidermal stem cells grew like paving stone after 5 days after the preparation of epidermal stem cells by enzyme digestion. The epidermal stem cells were purified by the rapid adhesion of epidermal stem cells to type IV collagen. When cultured at room temperature and cold digestion, the epidermal stem cells were digested by room temperature digestion and cold digestion. The results showed that the best digestion time was 6 h for cold digestion and 5 min for room temperature digestion. The positive colonies of epidermal stem cells were obtained by tissue block method and enzyme digestion method and passed to the 8th and 6th generation respectively. 2. Identification of rat epidermal stem cells. Morphologically, epidermal stem cells were small in size, round in shape, large in nuclear / cytoplasm ratio and able to form clones. Immunohistochemical staining showed that ESCs expressed p63C15, 尾 1-integrin and 伪 6-integrin.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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