一种培养原代小鼠角膜基质细胞的新方法

A novel method for culturing primary mouse corneal stromal cells in mouse

目的改良小鼠角膜基质细胞分离培养方法 ,为角膜基质细胞生物学和角膜组织工程等研究提供更好的种子细胞。方法采用Dispase酶消化法联合组织块贴壁法分离、培养原代小鼠角膜基质细胞,倒置显微镜下观察细胞生长情况,通过间接免疫荧光化学染色Vimentin表型和RT-PCR检测Vimentin、Lumican和CK12基因表达对培养的细胞进行鉴别。结果倒置显微镜下可见小鼠角膜基质细胞呈三角形和树枝状,细胞间连接明显,可形成网状连接;免疫荧光化学鉴定小鼠角膜基质细胞表达Vimentin阳性;RT-PCR显示Vimentin和Lumican基因表达阳性,CK12表达阴性。结论 Dispase酶消化法联合组织块贴壁法培养可获得具有角膜基质细胞特性的细胞,本方法为体外获得单纯小鼠角膜基质细胞提供了简单高效的途径。

Objective To improve the isolative and cultural ways of corneal stromal cells in mouse,and provide better seeding cells for the biological research and tissue engineering of corneal stromal cells.MethodsPrimary mouse corneal stromal cells were isolated and cultured by Dispase digestion and tissue pieces adherence.The growing characteristics of corneal stromal cells were observed under inverted microscope.The cultured cells were differentiated by immunochemically staining with Vimentin phenotypes and by RT-PCR for gene expression of Vimentin,Lumican and CK12.ResultsMouse primary corneal stromal cells showed triangle or dendritic morphology with many intercellular joints which could form networks under inverted microscope.Immunohistochemical method showed positive expression of Vimentin in corneal stromal cells.RT-PCR showed positive expression of Vimentin and Lumican genes and negative expression of CK12.ConclusionsCorneal stromal cells can be obtained by Dispase digestion and culture of stromal tissue pieces.This research establishes a simple and highly efficient way for culture of pure mouse corneal stromal cells in vitro.