目的:优化人外分泌汗腺细胞的分离方法,以高效地提取外分泌汗腺细胞。方法:新鲜的皮肤样本剪成组织微粒(大小约1 mm3),A组采用胰蛋白酶-乙二胺四乙酸(EDTA)和胶原酶Ⅱ型(2 mg/ml)体积分数1∶1混合的方法;B组采用传统消化方法,即胶原酶Ⅱ型;C组采用胰蛋白酶-EDTA消化的方法,三组同时置于恒温培养箱内,比较三种方法处理后汗腺细胞团的获取情况。将挑取的汗腺细胞团种于培养皿内,观察细胞贴壁和生长情况,并用流式细胞仪测定各组细胞的增殖指数,最后进行免疫细胞化学检测汗腺细胞标志性蛋白的表达。结果:A组和C组在消化30 min后,镜下可见少部分的汗腺细胞团,2 h后A组游离汗腺细胞团明显增多,C组游离汗腺细胞团很少;B组在消化6 h后,才出现部分游离的汗腺细胞团。将汗腺细胞团培养3 d后发现C组汗腺细胞贴壁情况差、成活细胞少;A组和B组的汗腺细胞贴壁情况良好,培养9 d后呈"铺路石样"生长,细胞增殖指数分别为(18±4)%和(17±6)%,无明显差异,免疫细胞化学结果表明:两组细胞癌胚抗原(CEA)和细胞角蛋白7(CK7)表达均为阳性。结论:胰蛋白酶-EDTA和Ⅱ型胶原酶联合消化法能明显缩短汗腺细胞的分离时间,且不影响细胞的活性和增殖特性。
Objective: To optimize the methods of isolating human eccrine sweat gland cells in vitro so as to get efficiently primary human sweat glands. Methods: The fresh and normal skin tissue was cut into pieces of microskin about lmm3 and the following 3 group digestion buffer was applied to isolated gland cells. The digestion buffer of group A was the eqnivoluminal mixture of Trypsin-Ethylene Diamine Te- tmacetic Acid(EDTA) and collagenase-Ⅱ (2 mg/ml). The digestion buffer of group B was collagenase-Ⅱ (2 mg/ml) traditionally and group C was Trypsin-EDTA. These three groups were placed into an incubator simultaneously and the emerging time of dissociated sweat glands was cal- culated. Sweat glands were sorted out and then placed in culture dish. The adherence and the growth of cells were observed. The proliferation index was detected by tlow cytometry. The identification of cultured cells was performed by immunocytochemical staining. Results. After di- gesting 30 rain in group A and C, a very few of dissociated sweat glands were emerging. But after digesting for 2 h , there were lots of dissoci- ated sweat glands emerging in group A rather than in group C. The emergence of dissociated sweat glands in group B would require at least 6 hours. After seeded in culture dishes, the sweat glands in group C couldn' t adhere to the wall of dish, but the sweat glands in group A and B adhered very well and even grew like paving stones after 9 days. In addition, the proliferation index were ( 18 ± 4) % and ( 17 ± 6) % respec- tively, there was no statistical difference. The results of immunocytochemical staining showed that the cells expressed carcino-embryonic antigen (CEA) and cytokeratin 7(CK7) in group A and B. Conclusion: Trypsin-EDTA combined with collagenase- Ⅱ can shorten the time of isolating sweat gland cells and have no effect on cell activity and proliferation.