体外培养不同代角膜缘干细胞特性分析

目的比较不同代角膜缘干细胞的生物学特性,为角膜缘细胞移植在临床中的氉应用提供依据。方法组织块培养法体外培养角膜缘干细胞,光镜观察、HE染色和免疫荧光染色分析原代、第1代和第2代角膜缘细胞形态学特性;流式细胞技术定量检测原代、第1代和第2代角膜缘细胞ABCG2表达;MTT比色法测定体外培养原代、第1代和第2代角膜缘干细胞的增殖活性变化。结果倒置相差显微镜下可见原代细胞培养24h有单个细胞从组织块边缘迁出,48h细胞局部可见拉网样生长趋势,72h出现大片细胞,细胞呈现多边形或多角形,96h细胞几乎铺满整个培养板。随着传代次数的增加,细胞形态变得不规则,CK3单克隆抗体表达阳性率逐渐增强;p63单克隆抗体原代和第1代细胞间表达阳性率无明显差别,第2代细胞表达量明显减弱。流式细胞检测显示原代、第1代和第2代细胞ABCG2表达阳性率分别为13.41%、22.96%和4.43%。MTT结果显示随传代次数增加细胞增殖活性逐渐下降。结论原代和第1代角膜缘干细胞可以作为制备组织工程角膜上皮细胞膜片的较理想种子细胞。

Characteristic analysis of corneal limbal stem cells with different passages in vitro

Objective To compare the biological characteristics of corneal limbal stem cells(LSC) with different passages,and provide the basis for LSC application in transplanation surgery.Methods LSC were cultured in vitro.Morphological characteristics of LSC with different passages(P0,P1 and P2) were compared by light microscopy,HE staining and immunofluorescence.The quantitative expression of ABCG 2 in LSC with different passages(P0,P1 and P2) was examined by flow cytometry,and the proliferative activity of LSC with different passages(P0,P1 and P2) was determined by MTT.Results At 24 hours,a few LSC migrated out from the cultured tissue edge;At 48 hours,the cells grew in mesh;At 72 hours,the cells grew in a sheet;At 96 hours,the cells grew in confluence.The morphology of LSC changed irregularly after several passaged.The number of CK3 positive cells increased,and the expression of p63 had no obvious difference between P0 and P1,however,it became weaker in P2;Flow cytometry test showed that the ABCG 2 positive expression in cell with P0,P1 and P2 were 13.41%,22.96% and 4.43%,respectively.MTT results demonstrated that the cellular proliferative activity gradually reduced after several passaged.Conclusion The LSC with P0 and P1 are suitable as ideal seed cells for making corneal epithelium grafts during corneal tissue engineering.