人角膜上皮干细胞的低钙培养及碱性成纤维细胞生长因子对其增殖的影响

A study on effect of basic fibroblast growth factor on human limbal stem cell proliferation cultured in low calcium medium

目的 探讨如何获取较纯的人角膜上皮干细胞 ,以及碱性成纤维细胞生长因子(fibroblastgrowthfactor,bFGF)对人角膜缘上皮干细胞增殖的影响 ,为干细胞的移植奠定基础。方法采用低钙培养法对人角膜缘组织行体外培养 ,将培养的细胞行AE5免疫荧光染色 ,观察培养细胞的分化状态 ,并将其分别置于不同浓度的bFGF中 ,通过数码照相技术和计算机图像分析系统 ,检测角膜干细胞的增殖。结果 体外培养的细胞以角膜上皮干细胞为主 ;不同浓度的bFGF(1～ 10 0ng/ml)可促进体外培养的人角膜缘上皮干细胞的增殖 (P <0 0 0 1) ,而各实验组间比较 ,差异无显著性(P >0 0 5 )。结论 将无钙培养液与低钙培养基结合 ,可获得较纯的、未分化的角膜干细胞 ;bFGF对人角膜上皮干细胞的增殖具有重要的促进作用。计算机图像分析系统适用于定量检测呈膜状生长的上皮细胞增殖 ,是一种新颖、实用、准确的检测手段。

Objective To study how to obtain relatively pure stem cells and the effects of bFGF on the human limbal stem cell proliferation in order to set up the foundation of cultured limbal stem cells for treatment of ocular surface and tear diseases. Methods Limbal button inoculation was used in low calcium culture. Monoclonal antibody AE5 was used to identify the stem cells. To examine the effects of bFGF (1 100 ng/ml) on stem cell proliferation, the technique of digital camera and computer image analysis system were used. Results Cultured cells were mostly corneal stem cells. This was the first time to combine the method of obtaining basal cell layer and culture with low calcium medium, which could harvest relatively pure and undifferentiated stem cells in localization and nature. The basic FGF (1 100 ng/ml) promoted cultured stem cells to proliferate significantly ( P <0 001). Conclusions Limbal stem culture provides resources of stem cells. It is very useful in treatment of ocular surface and tear diseases. The basic FGF plays an important role in proliferation of stem cells in graft. Computer image analysis system is an effective and simple way for evaluating the proliferation of a primary culture.