本文選題：表皮干細(xì)胞　切入點(diǎn)：分離培養(yǎng)　出處：《西北農(nóng)林科技大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 表皮干細(xì)胞(Epidermal Stem Cells,ESCs)是一類存在于皮膚組織中的多能性細(xì)胞,能夠自我更新且具有多相分化潛能。本研究分別取成年SD大鼠和新生大鼠的背部皮膚,分離培養(yǎng)ESCs,并用IV型膠原進(jìn)行純化,最終獲得ESCs,并對(duì)其生物學(xué)特性進(jìn)行了研究。通過本研究,優(yōu)化了大鼠表皮干細(xì)胞分離培養(yǎng)體系和鑒定方法,對(duì)建立大鼠表皮干細(xì)胞系有十分重要的現(xiàn)實(shí)意義,為下一步研究大鼠表皮干細(xì)胞轉(zhuǎn)基因及分化奠定基礎(chǔ)。本試驗(yàn)的具體內(nèi)容為: 1.大鼠表皮干細(xì)胞的分離培養(yǎng)及純化 取大鼠背部皮膚,分別采取組織塊法和酶消化法分離培養(yǎng)大鼠ESCs。組織塊法制備細(xì)胞時(shí),將皮膚剪成0.1cm×0.1cm的小塊,基底面向下貼于培養(yǎng)皿中,添加培養(yǎng)液并隔天換液。組織塊法制備表皮干細(xì)胞時(shí),3~4d后從組織塊邊緣長(zhǎng)出上皮樣細(xì)胞,7~12d后細(xì)胞呈鋪路石樣生長(zhǎng),在上皮樣細(xì)胞層上有小圓形細(xì)胞聚集呈集落樣生長(zhǎng)。培養(yǎng)至20 d,形成許多100~200個(gè)細(xì)胞組成的大克隆。酶消化法制備細(xì)胞時(shí)分別用兩種酶消化組織塊:2.0U/ml的中性蛋白酶消化過夜;0.25%胰酶+0.02%EDTA消化10min。酶消化法制備表皮干細(xì)胞時(shí),4~5d后細(xì)胞呈鋪路石樣生長(zhǎng),培養(yǎng)至12~15d,形成克隆,但是死細(xì)胞較多。利用表皮干細(xì)胞對(duì)IV型膠原的快速黏附特性純化表皮干細(xì)胞。傳代培養(yǎng)時(shí),采用室溫消化法和冷消化法消化傳代,結(jié)果證明冷消化5～6h為最佳消化時(shí)間而室溫消化最佳時(shí)間為5min。組織塊法和酶消化法均能得到表皮干細(xì)胞陽(yáng)性集落且分別傳至第8代和第6代。 2.大鼠表皮干細(xì)胞的鑒定 從形態(tài)上觀察,表皮干細(xì)胞體積較小、圓形,核質(zhì)比大,能夠形成克隆。免疫組化染色結(jié)果顯示,ESCs表達(dá)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 偽 6-integrin.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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本文編號(hào)：1577899