家兔(Oryctolagus curiculus)角膜内皮细胞系的建立及其生物相容性研究
发布时间:2018-03-22 16:00
本文选题:家兔 切入点:角膜内皮细胞 出处:《中国海洋大学》2005年硕士论文 论文类型:学位论文
【摘要】:角膜内皮盲是一种常见的角膜病,尽管可以通过角膜移植来治愈,但由于角膜的捐献数量极其有限和患者的排斥反应而使许多患者无法重见光明。人工角膜组织工程技术是当今治疗角膜内皮盲等的研究热点,其亟需解决的关键问题之一是如何获得足量的角膜内皮细胞。而角膜内皮细胞的规模化体外培养是获得大量角膜内皮细胞的理想途径,也是人工角膜的重要研究内容之一,对于角膜病理生理学、药理毒理学、免疫学、分子生物学研究均具有重要意义。但到目前为止,仍没见到有关家兔角膜内皮细胞未经转染而成功建系的报道。为了探索哺乳动物角膜内皮细胞的建系技术,以便为组织工程化人工角膜提供足量的细胞材料,本文以家兔角膜内皮为材料,对角膜内皮细胞的体外培养、细胞系的建立及其鉴定展开了研究。 为了成功启动家兔角膜内皮细胞的体外培养,本文取活家兔的角膜,用氯化汞溶液和庆大霉素依次进行消毒,经0.25%胰蛋白酶液部分处理角膜内皮层后,将角膜内皮面朝下贴于24孔培养板中,补加含有硫酸软骨素、硫酸软骨素氧化降解物、羧甲基壳多糖、N-乙酰葡萄糖盐酸盐、氨基葡萄糖盐酸盐、家兔角膜基质细胞培养液、牛眼生素、EGF和bFGF的DMEM-F12培养液(20%胎牛血清),置入37℃、5%CO_2培养箱中培养。为了获得纯净的角膜内皮细胞,每隔12~48小时进行揭膜和转孔再培养,进而成功启动了家兔角膜内皮细胞的原代培养。原代培养中的角膜内皮细胞多为四角形或多边形,一周后逐渐伸展成为多角形的内皮样,三周后细胞长成单层,此时细胞饱满、透光性好。待角膜内皮细胞长成单层后,便采用胰蛋白酶消化法进行传代培养。传代培养中的细胞,开始时绝大多数为多角形内皮样形态,但随着传代次数增加,逐渐转变为内皮样与成纤维样两种细胞形态共存的生长状态。传至第49代的角膜内皮细胞,主要呈现为成纤维样细胞形态,其生长与分裂状况良好。此时,细胞群体的倍增时间为2.24天,
[Abstract]:Corneal endothelium blindness is a common corneal disease, although it can be cured by corneal transplantation. However, because of the extremely limited amount of corneal donation and the rejection of patients, many patients can not see the light again. Tissue engineering technology of artificial cornea is a hot research topic in the treatment of corneal endothelium blindness. One of the key problems that need to be solved is how to obtain enough corneal endothelial cells, and the in vitro culture of corneal endothelial cells is an ideal way to obtain a large number of corneal endothelial cells, which is also one of the important research contents of corneal prosthesis. For corneal pathophysiology, pharmacology, toxicology, immunology, molecular biology, but so far, In order to explore the technique of establishing corneal endothelial cells in mammals in order to provide enough cellular materials for tissue engineered cornea, there is still no report about the successful establishment of rabbit corneal endothelial cells without transfection. In this paper, rabbit corneal endothelium was used as the material to study the culture, establishment and identification of corneal endothelial cells in vitro. In order to successfully initiate the rabbit corneal endothelial cell culture in vitro, the rabbit cornea was harvested and disinfected with mercuric chloride solution and gentamicin in turn. The corneal endothelium was partially treated with 0.25% trypsin solution. The corneal endothelium was pasted face down to the 24-well culture plate, supplemented with chondroitin sulfate, chondroitin sulfate oxidative degradation, carboxymethyl chitosan N-acetyl glucose hydrochloride, glucosamine hydrochloride, rabbit corneal stromal cell culture medium. In order to obtain pure corneal endothelial cells, 20% fetal bovine serum was cultured in 37 鈩,
本文编号:1649359
本文链接:https://www.wllwen.com/yixuelunwen/binglixuelunwen/1649359.html
最近更新
教材专著