表皮干细胞抽提物重编程脂肪干细胞表达表皮细胞表型的实验研究

Research of Reprogramming of Adipose Derived Stem Cells to Express Keratinocyte Phenotype Using Cell Extracts of Keratinocyte Stem Cells

目的研究富集的表皮干细胞(Keratinocyte enriched with epidermal stem cells,KSC)抽提物对重编程人脂肪干细胞(Adipose derived stem cells,ASCs)表达表皮细胞表型的影响。方法常规方法收集表皮细胞(Keratinocyte,KC)后,应用Ⅳ型胶原差速贴壁法分别收集KSC与富集后剩余的表皮细胞(Keratinocyte enriched with epidermal stem cellsleft,KCL),鉴定K19和P63的阳性表达率,Gimsa染色法测定KC、KSC、KCL的克隆形成率,分别制备KC、KSC、KCL的细胞抽提物,作用于链球菌溶血素-O(SLO)通透处理过的原代ASCs,分别应用流式细胞仪与Western-blot测定重编程后ASCs中广谱角蛋白(Pan cytokeratin,P-CK)与ASCs的BRG1表达变化。结果 KSC与KC、KCL来源的细胞抽提物重编程ASCs的CK及BRG1表达率,均有明显统计学差异(P<0.01)。结论脂肪干细胞在表皮细胞抽提物的诱导作用下能表达表皮细胞表型,且对表皮细胞优化处理后的KSC有更加显著的重编程作用。

Objective To evaluate the role of the cell extracts of keratinocyte stem cells（Keratinocyte enriched with epidermal stem cells,KSC） express keratinocytes phenotype of reprogramming of the adipose derived stem cells（Adipose derived stem cells,ASCs）.Methods Keratinocyte（KC） were collected in conventional methods,then KSC were isolated from KC with differential attachment to recycled collagen Ⅳ and KCL were gathered by not attached cells in suspension culture solution.Clone forming efficiency（CFE） of KC,KSC,KCL were detected with Gimsa staining methods.Cell extracts of KC,KSC and KCL were prepared respectively.Primary cultured adipose derived stem cells were permeabilized using streptolysin O（SLO）,and then incubated with cell extracts.Flow cytometry and western-blot were used to detect the expression of pan cytokeratin（Pan cytokeratin,CK） and Brahma-related Gene 1（BRG1） of reprogrammed ASCs.Results Cell extracts of KSC comparing with that of KC and KCL,had a statistically significant difference（P＆lt;0.01）in reprogramming of ASCs with high expression of CK and BRG1.Conclusion Adipose derived stem cells could be induced to express keratinocyte phenotype with cell extracts of keratinocytes,and optimized keratinocyte stem cells have superiority of reprogramming ability.