分离培养汗腺导管部细胞的新方法

A new way for isolation and cultivation of sweat gland ductual cells from human split-thickness skin in vitro

目的探讨建立汗腺导管部细胞分离的新技术。方法成人仞厚皮片和薄中厚皮片标本（n=10）剪碎后用Ⅱ型胶原酶消化12h，吸取并转移汗腺导管到培养皿中贴壁培养。应用流式细胞仪、免疫组织化学染色和逆转录．聚合酶链式反应（RT—PCR）以及蛋白印迹（Western Blot）分析检测培养细胞的汗腺特异标志CEA、CK8、CK18、CK19抗原表达，并用膜片钳技术检测培养细胞膜上阿米洛利（amiloride）敏感Na^＋离子通道，用t检验比较分析两组间实验数据。结果汗腺导管贴壁48h后，围绕汗腺导管出现单层扁平的上皮细胞，生长2～4周融合成片。流式细胞学检查示原代培养汗腺导管细胞与原代培养汗腺细胞在癌胚抗原（CEA）阳性率[（90．26±1．12）％帆（89．70±1．43）％]和细胞角蛋白8（CK8）阳性率[（94．41±1．84）％憾（93．65±1．63）％]上，差异无统计学意义（P〉0．05）。形态学染色汗腺导管细胞抗CEA、CK8、CK18、CK19染色均为阳性。RT—PCR表明原代培养汗腺导管细胞表达CEA、CK8、CK18、CK19基因，Western Blot清晰显示CEA条带，CK8、CK18、CK19蛋白条带。膜片钳检测表明原代培养汗腺导管细胞膜上存在amiloride敏感Na^＋离子通道。无血清表皮细胞EpiLife培养基在汗腺导管细胞生长过程中抑制成纤维细胞生长。结论从仞厚皮片和中厚皮片分离培养汗腺导管部细胞的方法较传统的分离方法具有简便快速的优点，EpiLife培养基可抑制培养过程中成纤维细胞的生长，可以在体外建立最佳汗腺导管细胞模型。

Objective To explore a new method of isolation and culture of eccrine sweat gland ductual cells from human split-thickness skin graft in vitro. Methods Human split-thickness skin graft which was presented by volunteer （n = 10） was digested with type Ⅱ collagenase, and then sweat gland duct were isolated from the split-thickness skin graft, primary cultures were incubated at 37℃ in humidified atmosphere of 5% CO2,95% 02. The cultured eccrine sweat gland ductual cells were identified by analysis CEA, CKS, CK18, CK19 antigens expression with flow cytometry, RT-PCR and Western Blot, and by detecting the electrophysiology with whole cell patch clamp technology. Results The isolated eccrine sweat gland ductual cells could grow by adhering to the wall, proliferate in vitro after 48 h of adhering to the wall, and confluens after 2-dweeks of adhering to the wall. The FACs analysis showed the expression of CEA was （90.26± 1.12）%, （89.70＋ 1. 43）%, and CK8 was （94.41 ±1.84）%, （93.65± 1.63）% in primary cultured sweat gland ductual cells and primary cultured eccrine sweat gland cells, repeetively, and there is no significant difference between the two groups （P 〉 0. 05）. Immunocytochemistry staining showed CEA,CK8, CK18, CK19 was positive in sweat gland duct cells, RT-PCR revealed that CEA, CKS, CK18 and CK19 gene expression in sweat gland ductual cells, and Western Blot analysis showed the expression of CEA brand,CK8 brand,CK18 brand, and CK19 brand in sweat gland ductual cells, patch clamp indicated that this cells has distinct amiloride sensitive Na^＋ channels. Conclusions The cultured human eccrine sweat gland duet cells in vitro display the markers and biological characteristics of sweat gland epithelial lineage, and this method of digest the split-thickness skin graft to get the sweat gland duct cells is better than classical dissect sweat gland under dissect microscope.