胰酶-EDTA差速消化法纯化原代培养的汗腺细胞  

Purification of primary cultured sweat gland cells by differential digestion with trypsin-EDTA

作　　者：谭志军[1] 陈艳[2] 赵洪良[2] 赵换军[1] 孙同柱[3] 马奎[3] 张翠萍[3] 付小兵[3] 

机构地区：[1]天津医科大学,天津300070 [2]解放军总医院,北京100853 [3]解放军总医院第一附属医院全军创伤修复与组织再生重点实验室,北京100048

出　　处：《感染．炎症．修复》2013年第4期225-227,I0002,共4页Infection Inflammation Repair

基　　金：国家973计划资助项目(2012CB518105);国家自然科学基金项目(81121004;81230041;81171798)

摘　　要：目的：探讨胰酶-EDTA差速消化法纯化原代培养的汗腺细胞的效果.方法：取新鲜的腹部手术患者腹部皮肤,D-Hanks液浸洗去除皮肤中的血液,剪成1 mm×1 mm大小的皮粒,将剪碎的皮粒放入装有5 ml （2 mg/ml） 的Ⅱ型胶原酶的培养皿中,置于37 ℃、5%CO2孵箱中消化8-12 h,倒置相差显微镜直视下吸取游离的汗腺,用汗腺培养基培养5-7 d,观察细胞形态和生长状况,加入胰酶-EDTA混合溶液2 ml（胰蛋白酶0.25,EDTA 0.53 mol/L）消化3 min,成纤维细胞收缩变圆后立即加入胎牛血清5 ml终止消化,吸弃消化液,D-Hanks冲洗2~3次,加入5 ml含10%胎牛血清DMEM培养液继续培养.镜下观察成纤维细胞去除效果,并对纯化后的细胞进行免疫细胞化学鉴定.结果：汗腺分离培养5-7 d后,汗腺细胞呈铺路石样生长,并且汗腺细胞周围生长有大量的成纤维细胞;用胰酶-EDTA消化3 min后,细胞计数显示约有95%以上的成纤维细胞被去除;免疫细胞化学鉴定结果显示纯化后的细胞CEA和CK8染色阳性,而波形蛋白和成纤维细胞特异性蛋白染色阴性.结论：胰酶-EDTA差速消化法能够简单、快速、高效地纯化汗腺细胞.Objective ： To investigate the effects of differential digestion with trypsin-EDTA on purification of primary cultured sweat gland cells. Methods：Full thickness skin was collected from patients undergoing laparotomy after informed consent, and blood was rinsed off with 5 ml D-Hanks solution. The skin was cut into pieces of about 1 mm × 1 mm insize, and digested for 8 to 12 hours with 5 ml （2 mg/ml） collagenase- II in an incubator with 5 % CO2 at 37 ℃. Sweat glands were isolated under an inverted phase contrast microscope and cultured with 5 ml culture medium for 5 to 7 days, and the morphology and growth of sweat gland ceils were observed. Then, the ceils were digested for 3 minutes with 2 ml of 0. 25 % trypsin-0.53 mol/L EDTA solution. Fetal bovine serum （5 ml） was added to terminate the digestion when the round fibroblasts were identified under the microscope. The digesting medium, was discarded, and the ceils were washed with D-Hanks solution for 2 or 3 times, and they were cultured with DMEM culture medium containing 10% fetal bovine serum. The effect of fibroblast removal was evaluated under microscope. The purified cells was identified by immunocytochemical staining. Results：Sweat gland cells grew like cobble＆stones after being cultured 5 to 7 days. There was a large number of fibroblasts around sweat gland cells. After digesting for 3 minutes with Trypsin-EDTA, more than 95% of fibroblasts were removed. The results of immunocytochemical staining showed that the purified cells expressed CEA and CKS, but not vimentin and fibroblast specific protein. Conclusions：Differential digestion with Trypsin-EDTA can remove fibroblasts from culture system of sweat gland cells, rapidly and effectively.

关 键 词：胰酶-EDTA汗腺 分离 纯化 成纤维细胞 

分 类 号：R622.3[医药卫生—整形外科]