本文關(guān)鍵詞： 羊膜 角膜緣上皮細(xì)胞 培養(yǎng)　出處：《大連醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 研究背景:眼表疾病是眼科常見病,其中許多疾病都存在角膜緣干細(xì)胞缺陷或功能障礙,治療也相當(dāng)棘手�；诮悄ぞ壐杉�(xì)胞的新概念,近10年來眼表面重建研究集中于角膜緣部上皮細(xì)胞,成為當(dāng)今國際眼科研究熱點(diǎn)之一。自體角膜緣上皮移植已在臨床上廣泛開展并取得良好的療效。然而,由于自體取材在很多情況下受限,同種異體角膜緣上皮移植和以體外培養(yǎng)的角膜緣干細(xì)胞移植是最新的研究目標(biāo)。 目的:建立以羊膜為載體兔角膜緣上皮細(xì)胞的體外培養(yǎng)方法,了解其生物學(xué)特性,為最終實現(xiàn)以體外培養(yǎng)的兔角膜緣干細(xì)胞移植提供實驗依據(jù)。 方法:選擇新鮮的兔眼球,在凈化臺內(nèi),用含有“雙抗”的生理鹽水進(jìn)行沖洗后,制備兔角膜緣組織,將其剪切成約1mm×1mm大小的組織塊。以去除上皮細(xì)胞的羊膜為載體,采用三種方法對兔角膜緣上皮細(xì)胞(limbal epithelial cell ,LEC)進(jìn)行接種培養(yǎng),分別為:組織塊直接接種培養(yǎng)法、組織塊制備單細(xì)胞培養(yǎng)法、組織塊經(jīng)酶消化處理后培養(yǎng)法。培養(yǎng)基分別采用了RPMI-1640和DMEM+Ham-F12,并添加了相同的營養(yǎng)因子。倒置顯微鏡下觀察培養(yǎng)細(xì)胞體外生長的形態(tài)。 結(jié)果:1、組織塊直接接種培養(yǎng)法:接種培養(yǎng)第3天,上皮細(xì)胞開始從角膜緣組織塊周圍移行到羊膜上,形成典型的“沙灘樣”移行帶。第5～7天,大部分組織塊上的細(xì)胞從移行帶向外生長形成生長暈,相互連接成膜狀。第13～15天,細(xì)胞匯合在一起,排列緊密,鋪滿整個羊膜。2、組織塊制備單細(xì)胞培養(yǎng)法:培養(yǎng)接種5天后上皮細(xì)胞開始在羊膜上貼壁,細(xì)胞呈圓形,大小一致。第8天左右細(xì)胞開始生長,2周時細(xì)胞匯合在一起,形成網(wǎng)狀結(jié)構(gòu)。同時,生長的上皮細(xì)胞中有成纖維樣細(xì)胞的混雜生長,但各自保持其良好的細(xì)胞形態(tài)。3周左右形成良好的單層,細(xì)胞生長呈膜片狀擴(kuò)展,整個羊膜之上鋪滿細(xì)胞,有明顯的擴(kuò)展界限。但羊膜之上有兩種細(xì)胞混合生長,成纖維細(xì)胞的生長明顯強(qiáng)于上皮細(xì)胞。3、組織塊經(jīng)酶消化處理后培養(yǎng)法:培養(yǎng)第7天,部分上皮細(xì)胞開始在羊膜上貼壁,但大多以散在形式生長。在部分細(xì)胞貼壁的同時,培養(yǎng)液中有懸浮生長的細(xì)胞,換液后仍然存在。第15天,羊膜上貼壁的細(xì)胞逐漸增多,但大多數(shù)以單細(xì)胞生長,也可見少量的細(xì)胞團(tuán)。第20～25天左右,培養(yǎng)的細(xì)胞匯合在一起,形成團(tuán)狀結(jié)構(gòu),逐漸成網(wǎng)狀后連接成片,但不能形成良好的單層。 結(jié)論:1、羊膜是兔LEC體外培養(yǎng)的良好載體。2、在以羊膜為載體的三種接種培養(yǎng)方法中(①組織塊直接接種培養(yǎng)法;②組織塊制備單細(xì)胞培養(yǎng)法;③組織塊經(jīng)酶消化處理后培養(yǎng)法),組織塊直接接種培養(yǎng)法效果最佳。3、以RPMI-1640和DMEM+ Ham-F12(體積比3:1)為培養(yǎng)基培養(yǎng)兔LEC,在培養(yǎng)基中添加相同的營養(yǎng)成份,培養(yǎng)結(jié)果無明顯差異。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

【引證文獻(xiàn)】

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1 梁曄;海藻多糖生物交聯(lián)劑的制備、性質(zhì)及其生物學(xué)性能研究[D];中國海洋大學(xué);2008年

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本文編號：1442306