去除型人皮肤成纤维饲细胞系的建立及其重建角膜表层的研究

Development of genetically modified eliminable human dermal fibroblast feeder cells for ocular surface regeneration medicine

目的探讨去除型人皮肤成纤维饲细胞系的建立及其重建角膜表层的相关研究。方法将增强型绿色荧光蛋白(EGFP)、端粒酶逆转录酶(hTERT)和单纯疱疹病毒胸苷激酶(HSV-TK)3种基因导入人皮肤成纤维细胞,建立荧光标记的永生化的可去除型TERT+TK-D人源饲细胞系。将创建的细胞系经丝裂霉素C处理后作为饲养细胞与人角膜缘干细胞共培养,并与3T3饲细胞的培养结果作比较。结果TERT+TK-D细胞系在体外经过6个月的连续传代后仍然表达绿色荧光蛋白,保持旺盛的分裂能力,对更昔洛韦敏感。TERT+TK-D组的克隆形成率(colony forming efficiency,CFE)为(11.77±0.21)%,与3T3组CFE(12.8±1.61)%比较,差异无统计学意义(P=0.332);经过共培养,两组都形成了4~5层复层上皮细胞片。角膜缘干细胞克隆和上皮细胞片的免疫荧光染色及Real-time PCR定量分析结果显示,TERT+TK-D组角蛋白K3表达低于3T3组;PCR结果证实TERT+TK-D饲细胞在更昔洛韦作用下凋亡、裂解,没有混入培养获得的角膜上皮细胞片中。结论转基因荧光标记的永生化的去除型人皮肤成纤维饲细胞有望替代3T3细胞用于角膜再生医疗。

Objective To explore the establishment of a line of labeled immortalized eliminable human dermal fibroblast cells for ocular surface regeneration medicine.Methods The enhanced green fluorescent protein(EGFP)gene,human-derived telomerase reverse transcriptase(hTERT)gene,and herpes simplex virus thymidine kinase(HS-TK)gene were transfected into human dermal fibroblast cells to establish labeled immortalized eliminable feeder cells.Established genetically modified dermal fibroblasts(TERT+TK-D)were treated with mitomycin,co-cultured with human limbal stem/progenitor cells to regenerate epithelium sheets in order to compare with 3T3 feeder cells.Results Established TERT+TK-D feeder cells can maintain immortalization,visualization,and eliminable characteristics during 6 months of continuous passages,and they were sensitive to ganciclovir.The colony-forming efficiency(CFE)of limbal stem/progenitor cells was similar in the TERT+TK-D group(11.77±0.21)%and the 3T3 group(12.8±1.61)%(P=0.332).All cell sheets were well stratified into four to five layers.The TERT+TK-D group colonies and epithelial cell sheets showed weaker staining of corneal epithelium differentiation marker K3 than the 3T3 group via limbal stem cell clones,immunofluorescence staining of epithelial cell sheets and mRNA quantitative analysis.Moreover,PCR analysis against the long terminal repeat sequence of the lentiviral vector integrated into the genetically modified feeder cells showed no contamination of ganciclovir-treated regeneration epithelial sheets.Conclusions Genetically modified labeled immortalized eliminable human dermal feeder cells are promising substitutes for 3T3 feeder cells for xenogeny-free ocular surface regeneration.