人未成熟朗格汉斯细胞的分选及鉴定

Sorting and identification of human immature Langerhans cells

目的分离并鉴定健康人皮肤组织内未成熟状态的朗格汉斯细胞(LC)。方法把取得的人包皮组织剪成特定大小后采用分散酶Ⅰ(dispaseⅠ)消化过夜,分离表皮和真皮;Ⅰ型胶原蛋白酶在37℃消化表皮组织2 h并结合摇床振荡制成单细胞悬液,通过特异性的分子标志物,应用流式细胞仪分选获得人CD3^-CD14^-CD1a^+CD45^+LC,采用免疫荧光技术检测CD207的表达对其进行鉴定,并用台盼蓝染色检测其活力。结果分选得到未成熟LC,纯度为91. 31%,平均存活率为93. 16%。结论成功分选获得活力好、纯度高的未成熟人LC。

Objective To isolate the immature Langerhans cells(LCs) of human epidermis and to identify the phenotype based on their surface marker.Methods After treatment of piecemeal tissues of human foreskin with dispase Ⅰ digesting overnight,the separated epidermis was further digested by collagenase Ⅰ at 37℃ in a shaker for 2 hours to produce a suspension of dispersive epithelium,in which,LCs population of CD3^-CD14^-CD1a^+CD45^+ was harvested in flow cytometry influx with certain antibodies and identified further by immunofluorescence assay of anti-CD207 antibody as while the vitality of these ceils were observed with trypan blue staining.Results The population containing 91.31% of immature LCs was sorted and harvested,which showed 93.16% of survival rate.Conclusion Technical procedure of sorting immature LCs from human foreskin was established and ascertained being effective.