| 兔角膜缘上皮干细胞体外扩增及生物学鉴定 |
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| 引用本文： | 莫链杰,刘小玲,叶宇峰,柯莉芹,任王芳,张春芳,吴连宝,张方骅.兔角膜缘上皮干细胞体外扩增及生物学鉴定[J].中国神经再生研究,2011,15(1):174-178. |
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| 作者姓名： | 莫链杰 刘小玲 叶宇峰 柯莉芹 任王芳 张春芳 吴连宝 张方骅 |
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| 作者单位： | 杭州师范大学，临床医学院，浙江省杭州市 310036,杭州师范大学，医学实验中心，浙江省杭州市 310036,杭州市第一人民医院眼科，浙江省杭州市 310006,杭州师范大学，临床医学院，浙江省杭州市 310036,杭州师范大学，临床医学院，浙江省杭州市 310036,杭州师范大学，临床医学院，浙江省杭州市 310036,杭州师范大学，临床医学院，浙江省杭州市 310036,杭州师范大学，临床医学院，浙江省杭州市 310036 |
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| 基金项目： | 2008年浙江省大学生科技创新基金(ZX09030100)和杭州师范大学攀登工程基金(YS01210010)资助 |
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| 摘 要： | 背景：角膜缘干细胞体外培养的关键在于建立稳定的体外培养体系，包括角膜缘干细胞的定位、培养条件、载体选择和鉴别方法等。
目的：探索兔角膜缘上皮干细胞体外扩增方法，并对其生物学特性进行鉴定。
方法：采用兔角膜缘组织块培养法，以人羊膜为载体，在体外进行兔角膜缘上皮干细胞原代和传代培养。倒置显微镜观察其体外生长特征；苏木精-伊红染色以及扫描电镜观察其形态；AE5和P63单克隆抗体免疫组织化学染色鉴定其蛋白表达。
结果与结论：采用组织块培养法可在体外获得角膜缘上皮干细胞，能成功传代培养且保持较高增殖潜能。培养于去上皮羊膜上的干细胞可融合成片，呈“拉网”现象。原代角膜缘上皮干细胞AE5单克隆抗体染色阳性率低于5%，P63染色阳性达90%；随传代次数增加AE5染色阳性率增高，P63染色阳性率降低。结果显示兔角膜缘组织块培养法可以在体外成功获得角膜缘上皮干细胞，原代和传代细胞均具有干细胞特性，以羊膜为载体培养可形成角膜移植片。
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| 关 键 词： | 兔 角膜缘上皮干细胞 生物学特性 羊膜 角膜上皮移植片 |
In vitro amplification and biological characterization of rabbit corneal limbal epithelial stem cells |
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| Mo Lian-jie,Liu Xiao-ling,Ye Yu-feng,Ke Li-qin,Ren Wang-fang,Zhang Chun-fang,Wu Lian-bao and Zhang Fang-hua.In vitro amplification and biological characterization of rabbit corneal limbal epithelial stem cells[J].Neural Regeneration Research,2011,15(1):174-178. |
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| Authors: | Mo Lian-jie Liu Xiao-ling Ye Yu-feng Ke Li-qin Ren Wang-fang Zhang Chun-fang Wu Lian-bao and Zhang Fang-hua |
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| Institution: | Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China,Medical Research Center of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China,Department of Ophthalmology, Hangzhou First People s Hospital, Hangzhou 310006, Zhejiang Province, China,Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China,Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China,Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China,Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China,Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China |
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| Abstract: | BACKGROUND: How to establish a stable in vitro culture system, including location of corneal limbal epithelial stem cells, in vitro sample harvest, in vitro culture, vector selection, as well as identification methods, play a key role in corneal limbal epithelial stem cells culture.
OBJECTIVE: To culture the isolated rabbit corneal limbal epithelial stem cells and to identify the biological properties of cultured cells.
METHODS: The primary rabbit cornel limbal epithelial stem cells were isolated and cultured with tissue inoculation using human amniotic membrane as vector. The growth features of cells were observed under an inverted microscope. The morphology of cells was observed by hematoxylin-eosin staining and a scanning electron microscope. Furthermore, the monoclonal antibody AE5 and P63 two-step immunohistochemical staining were used to identify limbal epithelial stem cell protein expression.
RESULTS AND CONCLUSION: The rabbit corneal limbal epithelial stem cells could be successfully cultured and maintained a relatively high value-added potential in vitro. Rabbit corneal limbal epithelial stem cells cultured on the amniotic membrane pull netted cellular layer. The AE5 monoclonal antibody positive rate of primary cultured cells was about 5% and P63 monoclonal antibody positive up to 90%. AE5-positive rate increased and P63-positive rate decreased with the increase in the number of subculture. The rabbit limbal epithelial stem cells can be successful culture and amplified on human amniotic membrane in vitro by limbal tissue culture method. The cultured cells maintain the characteristics of corneal epithelial cells. The rabbit corneal limbal epithelial stem cells can form grafts on the amniotic membrane. |
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