摘要
目的探讨兔角膜缘干细胞的体外培养方法并观察其生物学特性.方法采用消化培养法,分2组培养,1组为消化并过滤后的角膜缘干细胞培养组,另1组为消化后未过滤的带组织块培养组,均用含20%胎牛血清的培养液培养,待细胞长成良好单层后消化传代,分别观察记录细胞在原代和传代培养时的生长特性并行HE染色观察.结果过滤组角膜缘干细胞在培养24 h后开始贴壁生长,7～10 d形成良好单层,细胞以圆形或椭圆形为主,核较大,呈原始细胞特征,传代培养时见细胞体积逐渐增大,核变小,成纤维细胞少见;带组织块培养组原代培养时与过滤组无明显差异,传代时则见成纤维细胞渐增多,至第3代时占主导并抑制了角膜缘干细胞的生长.结论采用消化培养法可成功培养出兔角膜缘干细胞,培养时应滤除组织块以减少成纤维细胞的影响.
Objective To investigate ex vivo culture technique of rabbit limbal stem cells and observe its biological characteristics. Methods By enzymatic digestion, the culture cells were divided 2 groups. One was the culture group of limbal stem cells after digestion and filtering, the other was the culture group of limbal stem cells with tissue pieces after digestion but not filtering. The 2 groups of cells were all inoculated in medium containing 20 % fetal bovine serum and subcultured after they formed a confluent layer. The growth characteristics of the cells were observed and recorded in primary culture and subculture. Moreover, the cells were stained by hematoxylin and eosin（HE） methods. Results The limbal stem cells in filtering group began to adhere and grow after 24 hours and formed a confluent layer in 7～10 days. The primary culture cells showed mainly round shape or oval-shape and big nuclei and were similar to archaeocyte. Their volume increased gradually and nuclei changed smaller, moreover, fibroblasts were hardly to see in subculture. The cells in tissue group were similar to those in filtering group in primary culture, but fibroblasts increased gradually in subculture and were dominant in the third passage and inhibited the growth of limbal stem cells. Conclusion Rabbit limbal stem cells can be cultured successfully by enzymatic digestion culture method in vitro, but they should be filtered to remove tissue pieces after digestion to diminish fibroblasts’ disturbance.
出处
《江西医学院学报》
2005年第6期5-7,F0002,共4页
Acta Academiae Medicinae Jiangxi
基金
[江西省科委社会发展攻关资助项目]
关键词
角膜缘干细胞
培养
生物学特性
limbal stem cells
culture
biological characteristics
